METHODS AND

TECHNIQUES IN
PATHOLOGY
 INTRODUCTION

IT IS ESSENTIAL TO LEARN THE METHODS AND

TECHNIQUES EMPLOYED IN PATHOLOGY, SO THAT
SUBSEQUENT ADVANCED KNOWLEDGE OF
PATHOLOGY MAY BE LEARNED SWIFTLY.
FOLLOWING ARE SOME OF THE IMPORTANT AND
COMMON METHODS, TECHNIQUES AND TOOLS USED
IN THE STUDY OF PATHOLOGY
 I) HISTOPATHOLOGY
(SURGICAL PATHOLOGY)
HISTOPATHOLOGY DEALS WITH THE GROSS AND
MICROSCOPIC EXAMINATION OF TISSUES. TISSUES
ARE RETRIEVED SURGICALLY FROM THE BODY AND
SENT TO THE HISTOPATHOLOGY LABORATORY FOR
THE PATHOLOGICAL EXAMINATION OF THE
SPECIMEN. THE REPRESENTATIVE TISSUE SECTIONS
SHOULD NOT BE MORE THAN 3-4 MM IN THICKNESS.
 THE SECTIONS ARE PLACED IN STEEL OR PLASTIC
CAPSULES, OR CASSETTES AND ARE SUBJECTED TO
THE FOLLOWING STEPS OF PROCESSING, BEFORE
THEY CAN BE EXAMINED UNDER A MICROSCOPE.
1. FIXATION: THE MAIN PURPOSE OF FIXATION IS TO
PRESERVE TISSUES, SO AS TO MAINTAIN THEIR
NATURAL STRUCTURE AS CLOSELY AS POSSIBLE
FOR MICROSCOPIC EXAMINATION, AS WELL AS
PRESERVATION FOR MUSEUM DISPLAY.
FORMALINE IS THE MOST COMMONLY USED
FIXATIVE FOR LIGHT MICROSCOPY
THE OTHER FIXATIVES USED IN PATHOLOGY ARE
GLUTARALDEHYDE (FOR ELECTRON MICROSCOPY)
 2. TISSUE PROCESSING:

THE FIXED TISSUE MUST BE PROCESSED INTO A

FORM IN WHICH IT CAN BE CUT INTO THIN
MICROSCOPIC SECTIONS.
THE MAIN PURPOSE OF PROCESSING IS TO REPLACE
THE WATER IN THE TISSUES WITH AN EMBEDDING
MEDIUM (USUALLY PARAFFIN WAX), WHICH GIVES IT
A FIRM CONSISTENCY, SO AS TO ENABLE THIN
SECTIONING FROM THE TISSUE.
TISSUE PROCESSING INVOLVES THE FOLLOWING
STEPS:
 A. DEHYDRATION

THE PURPOSE OF THIS STEP IS TO REMOVE WATER

AND FIXATIVES FROM THE TISSUES (DEHYDRATE),
AND REPLACE IT WITH A DEHYDRATING FLUID
(MOST COMMONLY ETHYL ALCOHOL), WHICH IS
SUBSEQUENTLY REPLACED PERMANENTLY BY WAX.
OTHER DEHYDRATING AGENTS ARE ACETONE,
METHYL ALCOHOL, AND ISOPROPYL ALCOHOL.
 B. CLEARING

THE NEXT STEP IS CLEARING IN WHICH THE

DEHYDRATING AGENT (ETHYL ALCOHOL) IS
REPLACED BY A MEDIUM IN WHICH PARAFFIN WAX IS
COMPLETELY SOLUBLE.
THIS STEP ALSO MAKES THE TISSUE TRANSPARENT,
HENCE CALLED CLEARING.
THE MOST COMMONLY USED CLEARING AGENT IS
XYLENE.
OTHER CLEARING AGENTS INCLUDE TOLUENE,
BENZENE, CHLOROFORM.
 C. IMPREGNATION

IN THE STAGE IN WHICH THE CLEARING AGENT

(XYLENE) IS FINALLY REPLACED BY MOLTEN
PARAFFIN WAX.
WAX IMPREGNATION HARDENS THE TISSUES,
MAKING THEIR HANDLING AND SECTIONING EASIER.
 D. TISSUE EMBEDDING AND
BLOCKING:
BLOCKS ARE CONVENTIONALLY PREPARED BY USING
METALLIC LEUCKHART’S L MOLDS.
TISSUE EMDEDING AND BLOCKING CAN ALSO BE
PERFORMED IN SPECIALIZED EQUIPMENT CALLED
TISSUE EMBEDDING STATION.
 3. MICROTOMY
ONCE THE TISSUES HAVE BEEN EMBEDDED
INTO BLOCKS, THEY MUST BE CUT INTO THIN
SECTIONS THAT CAN BE PLACED ON THE SLIDE.
ROTARY MICROTOME IS THE MOST COMMONLY
USED MICROTOME, IN WHICH THE KNIFE IS
FIXED AND THE TISSUE BLOCK IS MOVABLE,
AND CAN BE ADVANCED TOWARD THE BLOCK
THE IDEAL HISTOPATHOLOGICAL SECTIONS
SHOULD BE UNIFORM AND 3-4 MICRONS IN
THICKNESS.
THE THINNER THE SECTIONS, THE BETTER AND
CRISPER ARE THE MORPHOLOGY AFTER STAINING.
THE SECTIONS ARE FLOATED IN A WATER BATH
MAINTAINED AT A TEMPERATURE OF 5˚C -10˚C LESS
THAN THE MELTING POINT OF THE WAX (54˚C - 62˚C).
THESE SECTIONS ARE NOW GENTLY LIFTED ONTO
GLASS SLIDES PRECOATED WITH EGG ALBUMIN,
WHICH ENHANCE THE ADHERENCE OF THE SECTION
TO THE SLIDE.
 4. HEMATOXYLIN AND EOSIN
STAINING
BEFORE STAINING, THE PARAFFIN IS WAX IS REMOVED
FROM TISSUE BY RUNNING THE SLIDES THROUGH
XYLENE TO ALCOHOL FOLLOWED BY WATER.
HEMATOXYLIN AND EOSIN (H&E) IS THE UNIVERSALLY
USED STAIN FOR ROUTINE MICROSCOPY.
HEMATOXYLIN IS A NUCLEAR STAIN, WHICH IS
EXTRACTED FROM THE LOGWOOD OF A TREE –
HAEMATOXYLUM CAMPECHIANUM.
HEMATIN, WHICH IS THE ACTIVE INGREDIENT
OF THE STRAIN, WHICH IMPARTS THE BLUE
COLOR, IS PRODUCED BY OXIDATION.
EOSIN IS A XANTHENE DYE, WHICH STAINS THE
CYTOPLASM PINK.
 5. MOUNTING AND COVER
SLIPPING
BEFORE STAINTING, THE PARAFFIN WAX IS REMOVED
FROM TISSUE BY RUNNING THE SLIDES THROUGH XYLENE
TO ALCOHOL FOLLED BY WATER.
THE STAINED SECTION ON THE SLIDE IS MOUNTED IN A
MOUNTING MEDIUM(DISTRENE, PLASTICISE, XYLENE),
AND THEN COVERED WITH A FINE PIECE OF GLASS, COVER
SLIP.
MOUNTING PROTECTS THE TISSUE FROM BEING
SCRATCHED, PROVIDES A BETTER OPTICAL QUALITY FOR
VIEWING UNDER THE MICROSCOPE AND PRESERVES THE
TISSUE SECTION FOR YEARS.