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Introduction To Histology-1

This document provides an introduction to the field of histology. It discusses that histology is the microscopic study of normal tissues, while histopathology is the study of structural changes in tissues due to disease. The key techniques in histology include tissue fixation, processing, sectioning with a microtome, and staining. Specialized staining methods allow visualization of specific structures like basement membranes, connective tissues, muscles, and nerves. Both physical slide examination with a microscope and virtual examination with digital images are used in histological analysis.

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Zen Fredy
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138 views43 pages

Introduction To Histology-1

This document provides an introduction to the field of histology. It discusses that histology is the microscopic study of normal tissues, while histopathology is the study of structural changes in tissues due to disease. The key techniques in histology include tissue fixation, processing, sectioning with a microtome, and staining. Specialized staining methods allow visualization of specific structures like basement membranes, connective tissues, muscles, and nerves. Both physical slide examination with a microscope and virtual examination with digital images are used in histological analysis.

Uploaded by

Zen Fredy
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Introduction to Histology

• Histology: microscopic study of normal tissues of body

• Histopathology : study structural changes due to disease

• Histotechnology: processing & preparation of tissues


Histology

• Real – with slides & microscopes

• Virtual – with photomicrographs projected


Histological methods
Objective lenses
REFERENCES
• D R Singh, Principles & Techniques in Histology, Microscopy &
Photomicrography Bangalore: CBS Publishers

• Yogesh Sontakke, Principles of Histological Techniques,


Immunohistochemistry & Microscopy 1st edition Hyderabad:Paras;
2017

• Lakshminarayanan, Histological Techniques – a practical manual 2nd


edition Mumbai: Bhalani Publishing House; 2011

• Kanai Mukherjee Medical Laboratory Technology Volume III New


Delhi:Tata McGraw-Hill; 1989

• Microtomes and microtome knives – a review and proposed


classification Mohammed et al Annal Dent Univ Malaya 2012;19(2):43-
50

• Chemical and physical basics of routine formaldehyde fixation


Thavarajah et al J Oral Maxillofac Pathol 2012 Sep-Dec; 16(3):400-405
Preparation of slides
Steps

• Tissue procurement

• Tissue fixation

• Tissue processing (Dehydration,


Clearing, Infiltration)

• Embedding

• Microtomy

• Staining
Tissue procurement

Human / Animal

Cadaveric / Biopsy
TISSUE FIXATION
• “Selective preparation of cells or tissue components so that the
sample can withstand subsequent steps for histological studies”

• TYPES:
1. Immersion
2. Perfusion : electron microscopy
3. Vapour : blood smears
4. Coating : cytology
5. Freeze drying : -160◦C
6. Microwave
Some fixatives
i. Simple

Eg: Formaldehyde, Glutaraldehyde

ii. Compound

Eg: Bouin’s fluid, Zenker’s fluid


ii. Formalin
• 37 to 40% aqueous solution of HCHO
• Fixation time: 1-2days
• Forms covalent cross-links with

i. tissue proteins

ii. Osmium tetroxide


• Electron microscopy
• Cytoplasmic organelles
ii. Cytological – Intracellular components
• Nuclear – eg: Carnoy’s fluid
• Cytoplasmic- eg: Champy’s fluid
DECALCIFICATION
• “Process of removing calcium salts from tissue, making them amenable for
sectioning”

• Methods:
I. CHEMICAL Nitric Acid / Formic Acid
or Chelating agents – EDTA

II. ION-EXCHANGE RESIN

• Ammonium salts of sulphonated polystyrene resin


• Regeneration – dilute N/10 HCl
III. ELECTROPHORETIC METHOD

IV. ULTRASONIC METHOD


7.5% acetic acid
DEHYDRATION
• “Process of removing water from fixed tissues”
• Wax cannot penetrate tissue in presence of free water in tissue
• Done with ascending grades of alcohol (70%-100%)

DEHYDRATING AGENTS
i. Ethanol – miscible with water & clearing agent
ii. Isopropyl alcohol (99%)
iii. Acetone – rapid action
CLEARING
• Dehydrating agent is removed from tissues
• De-alcoholisation : raises refractive index of specimen &
makes it translucent
• Facilitates penetration of paraffin wax into tissue
i. Xylene:
ii. Chloroform
INFILTRATION
• Impregnation/ Infiltration: “removal of clearing agent from tissues by using
embedding media ”

• Paraffin oven / Incubator:


 50-65 ◦C
Embedding: “Solid block preparation from embedding media to

hold tissue firmly for section cutting ”

a) Paraffin
b) Gelatin
v. PLASTIC EMBEDDING
• Electron microscopy
a) Acrylic media – Methacrylate
b) Araldite/ epoxy resin
AUTOMATED TISSUE PROCESSOR
MICROTOME
• Freezing microtome

• Cold microtome/ Cryostat:

• Vibratory knife microtome:


• Laser microtome:
• Infrared laser

• Ultramicrotome : section thickness in nanometers


vi. Set microtome to 5 microns for cutting

vii. Turn flywheel & cut section slowly


& at fixed rate
vi. Pick ribbon with soft painting
brush No.0 or 1

vii. Transfer ribbon to warm water bath for flattening

viii. Cut the ribbon to desired portion


STAINS
a) Basic dye – Positively charged
• Nuclear stain
• Stains anionic groups
E g – Hematoxylin –
Haematoxylon compechianum
(“blood wood”)

b) Acid dye – Negatively charged


• Cytoplasmic stain
• Stains cationic groups
• E g – Eosin

c) Neutral dye –
stains both cationic & anionic structures
• E g - Neutral red
TYPES OF STAINING METHODS
a) Progressive staining
• Nucleus stained first & then the cytoplasm
• Tissue left in stain – reaches desired end-point

b) Regressive staining
• Tissue is deliberately overstained
• Differentiation done till end-point is reached

DIFFERENTIATION
• Destain basic dye with weakly acidic medium
• Haematoxylin with acidified alcohol

• Destain acid dye with weakly basic medium


• Eosin with 0 1-0 5% conc Ammonium hydroxide
EOSIN
• Xanthene group of dyes

• Derived from fluorescein

a) Eosin B (bluish)
• More soluble in alcohol
• 0 5% strength – dissolved in 70% alcohol
• Staining time – 2 to 3 minutes

b) Eosin Y (yellowish)
• More soluble in water
• 5% aqueous solution – dissolved in tap water
PROCEDURE
DEPARAFFINISATION
• Warm slides on hot plate
• Xylene

REHYDRATION
• Absolute alcohol:
• 90% alcohol
• 70% alcohol
• 50% alcohol
• 30% alcohol
• Rinse in distilled water
• Place in hematoxylin for 2-3 minutes
• Rinse in distilled water – 3 minutes
• Differentiate in 1%acid alcohol (dip)
(1% HCl in 70% ethanol)

• “Blue” the sections in tap water for 5-7 minutes


• Rinse in distilled water
• Counterstain in 0 5-1% eosin for 1 minute
• Rinse in distilled water

• DEHYDRATION
50% alcohol
70 % alcohol dip
90 % alcohol
Absolute alcohol
(2 changes)

• Xylene (2 changes) – dip

• Slide ready for mounting


MOUNTING
ii. Water insoluble or resinous medium
E g – Canada balsam
DPX (Distrene, Plasticizer, Xylene)
H& E Stained tissue
SPECIAL STAINS
BASEMENT MEMBRANE – PERIODIC ACID SCHIFF

• Stains reddish purple


CONNECTIVE TISSUE FIBRES - VAN GIESON’S STAIN

Collagen – deep red


Epidermis – bright lemon yellow
Muscle & nerve cells – olive green
ELASTIC FIBRES - VERHOEFF’S METHOD:
MUSCLE TISSUE - MASSON’S TRICHROME:

Nuclei– black
Cytoplasm, Muscle – red
Collagen – green
NERVOUS TISSUE

• Myelin sheath: Osmium tetroxide

• Nissl substance: Cresyl Violet

• Neurite: Bielschowsky’s silver

• Astrocytes: Cajal’s gold sublimate


HORSERADISH PEROXIDASE
• Enzyme label in immunohistochemistry
• Haematin – active site
• Enzyme tagged antibody reacts with substrate to
form a chromogen brown-red end product
Slide –view Drawing
Virtual histology

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