Professional Documents
Culture Documents
Histopathologic Techniques
Histopathologic Techniques
Teasing or Dissociation
Squash Preparation (Crushing)
Smear Preparation (Streaking, Spreading,
Pull Apart, Touch or Impression Smear
Frozen Section
FS indications
- rapid diagnosis (guide for intra-
FS limitations
Numbering
Fixation
Dehydration
Clearing
Impregnation
Embedding
Blocking
Trimming
Sectioning
Staining
Mounting
Labelling
Fixation
Fixation
Fixation
Fixation
Microwave Technique
Microwave advantages:
Microwave disadvantages:
Fixative
Cheap
Stable
Safe to handle
Kills quickly
Minimum tissue
shrinkage
Rapid & even
penetration
Types of Fixative
According to composition
- Simple Aldehydes, metallic fixatives
- Compound
According to action
- Microanatomical
- Cytological Nuclear & Cytoplasmic
- Histochemical
Simple Fixatives
Aldehydes
Formaldehyde
Glutaraldehyde
Metallic Fixatives
Mercuric Chloride
Chromate Fixatives
Lead Fixatives
Picric Acid
Acetic Acid
Acetone
Alcohol
Osmium Tetroxide /
Osmic Acid
Heat
Microanatomical Fixatives
10 % Formol Saline
10 % Neutral Buffered
Formalin
Heidenhains Susa
Formol Sublimate
(Formol Corrosive)
Zenkers
Zenker Formol
(Hellys)
Bouins
Brasils
Cytological Fixatives
Nuclear:
Flemmings
Carnoys
Bouins
Newcomers
Heidenhains
Cytoplasmic
Histochemical Fixatives
Formaldehyde
Methanol oxidized
Cheap, readily available, easy to prepare,
stable, compatible w/ stains, penetrates
tissues well, preserves fat, mucin,
glycogen, for tissue photography
Irritating fumes, prolonged fixation may
bleach tissues
Formaldehyde precautions:
Paraformaldehyde formation
Well ventilated room
Not neutralized if concentrated explosion
Buffered or neutralized by adding
magnesium carbonate/CaCO3 wide
mouth bottle
Bleaching prevented by changing formalin
10 % Formol Saline
Glutaraldehyde
For LM, EM
Adv vs. HCHO: more stable effect, less
tissue shrinkage, less irritating
Disadv: more expensive, slow penetration
Mercuric Chloride
Mercuric Chloride
Mercuric chloride
Dezenkerization
Chromate Fixatives
Lead Fixatives
Disadavantage:
Polarization causes glycogen granules
to move towards the poles / ends of cells
TCA
Acetone
Heat Fixation
Post Chromatization
Secondary Fixation
Washing out
Tap water
50 70 % alcohol
Alcoholic iodine
Fixation
Retarded by:
Large size
Mucus
Fat
Blood
Cold
Enhanced by:
Decalcification
Decalcification*
Decalcification
Acids
Chelating Agents
Ion Exchange Resins (Ammonium form of
polystrene resin)
Electrical Ionization (Electrophoresis)
Decalcification
Acids
Most common
Stable
Easily available
Cheap
Nitric, hydrochloric, formic, TCA,
sulfurous, chromic, citric acid
Most common
Fastest
Disadvantage: inhibits nuclear stain
combine with formaldehyde or alcohol
Aqueous nitric acid 10%, formol nitric acid,
Perenyis, Phloroglucin nitric acid
Nitric Acid
Nitric Acid
Rapid acting
Good nuclear staining
Less tissue destruction than 10% aqeuous
nitric acid
Use fume hood
Lessen yellow tissue discoloration by 5%
sodium sulfate or 0.1 % urea
Nitric Acid
Nitric Acid
HCl
Formic Acid