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