INTRODUCTION AND MOLECULAR COMPOSITION, PIECES OF
ORGANS SHOULD BE PROMPTLY AND
HISTOLOGY
• THE STUDY OF THE TISSUES OF THE BODY
AND HOW THESE TISSUES ARE ARRANGED TO
CONSTITUTE ORGANS.
• FROM THE GREEK WORD HISTO MEANING
“TISSUES” OR “WEB”. *WEB BECAUSE OF
INTERWOVEN FILAMENTS AND FIBERS, BOTH
CELLULAR AND NONCELLULAR, WITH
MEMBRANOUS LINING.
• IT INVOLVES ALL ASPECTS OF TISSUE
BIOLOGY. *WITH THE FOCUS ON HOW CELLS’
STRUCTURE AND ARRANGEMENT OPTIMIZE
FUNCTIONS SPECIFIC TO EACH ORGAN.
PREPARATION OF TISSUE FOR STUDY:
*THE MOST COMMON PROCEDURE USED IN
THE STUDY OF TISSUES IS THE PREPARATION
OF HISTOLOGICAL SECTIONS OR TISSUE
SLICES THAT CAN BE STUDIED WITH THE AID
OF THE LIGHT MICROSCOPE.
BASIC STEPS IN TISSUE PREPARATION FOR
HISTOLOGY:
• FIXATION
• DEHYDRATION
• CLEARING
• INFILTRATION OR
IMPREGNATION
• EMBEDDING, CASTING OR
MOLDING
• BLOCKING
• TRIMMING
• SECTIONING OR CUTTING
• STAINING
• MOUNTING
LABELING *IMMEDIATELY AFTER REMOVAL OF TISSUE
FIXATION
• THIS METHOD CAN BE DONE BY CHEMICAL
OR LESS FREQUENTLY PHYSICAL METHODS.
• TO AVOID TISSUE DIGESTION BY ENZYMES
PRESENT WITH THE CELLS (AUTOLYSIS) OR BY
BACTERIA AND TO PRESERVE THE STRUCTURE
ADEQUATELY TREATED BEFORE, OR AS SOON
AS POSSIBLE, AFTER REMOVAL FROM THE
BODY.
• PRIMARY AIM: PRESERVE
MORPHOLOGY OF CELL
• SECONDARY AIM: HARDEN TISSUE
TO PROTECT FROM TRAUMA
• FORMALDEHYDE AND GLUTARALDEHYDE –
WIDELY USED FIXATIVE.
• FORMALDEHYDE: BEST GENERAL
FIXATIVE / BEST FOR IRON
CONTAINING TISSUES
• FORMALIN – ONE OF THE BEST FIXATIVES
FOR ROUTINE LIGHT MICROSCOPY. *A
BUFFERED ISOTONIC SOLUTION OF 37%
FORMALDEHYDE.
• TISSUES ARE PLACED IN FIXATIVES
ASAP AFTER REMOVAL FROM THE
BODY
MAIN FACTORS INVOLVED IN FIXATION
• PH: 6-8 OPTIMUM
• TEMPERATURE: GENERALLY, AT RT
• ELECTRON MICROSCOPY &
HISTOCHEMISTRY: 0-4C (TO
PREVENT DENATURATION OF
ENZYMES AND PROTEINS)
• MAST CELLS: RT
• NUCLEIC ACIDS: HIGHER TEMP
(RNA: 45C/ DNA: 65C)
• THICKNESS OF SECTION: 2-4mm
• CONCENTRATION: 10% FORMALIN
PRACTICAL CONSIDERATIONS:
• SPEED: ASAP
• PENETRATION: FORMALIN 1mm/hr
• VOLUME: 20X
DECALCIFICATION
• COMPLETE REMOVAL OF CALCIUM SALTS
FROM THE TISSUE FOLLOWING FIXATION
 • RECOMMENDED FLUID TO TISSUE RATIO:
20:1
• IDEAL TIME REQUIRED: 24-48 HRS (2-3 DAYS)
• AT 55C, TISSES WILL UNDERGO COMPLETE
DIGETSION WITHIN 24-48 HRS.
• THE HIGHER THE TEMP, FASTER
DECALCIFICATION AND HIGH RISK
OF DAMAGING TISSUE
TYPES OF DECALCIFYING AGENTS:
• ACID
• NITRIC ACID / HYDROCHLORIC
ACID / FORMIC ACID
• *TISSUE SOFTENERS: FOR UNDULY
HARD TISSUES WHICH MAY
DAMAGE THE MICROTOME KNIVES
• 4% PHENOL / 2%HCL
DEHYDRATION
• MOST OF THE WATER ARE REMOVED
• PROCESS OF REMOVING EXTRACELLUALR
AND INTRACELLULAR WATER FROM TISSUE
FOLLOWIUNG FIXATION AND PRIOR TO WAX
INFILTRATION
• THIS IS COMMONLY CARRIED OUT BY
IMMERSING SPECIMENS IN A SERIES OF
ETHANOL SOLUTIONS INCREASING THE
CONCENTRATION. *UNTIL PURE, WATER-
FREE ALCOHOL IS REACHED. WHICH
EFFECTIVELY REMOVES ALL WATER FROM
THE TISSUE.
• MOST COMMON: ALCOHOL
• ETHANOL: FOR ROUTINE
DEHYDRATION: EST DEHYDRATING
AGENT
• METHANOL
TYPICAL DEHYDRATION SEQUENCE FOR SPECIMENS: *
NOT MORE THAN 4mm THIICK
• 70% ETHANOL – 15 MINS
• 90% ETHANOL – 15 MINS
• 100 % ETHANOL – 15 MINS
• 100 % ETHANOL – 15 MINS
• 100 % ETHANOL – 30 MINS
• 100 % ETHANOL – 45 MINS
APPLICATION OF CLEARING:
*TO MAKE TISSUE, EMBRYOS, PARASITES
TRANSPARENT.
* FOR DEALCOHOLIZATION OF TISSUE
PREPARATORY TO WAX IMPREGRATION
CLEARING
• THE PROCESS WILL DISPLACE THE ETHANOL
IN THE TISSUE. *THE CLEARING SOLUTION IS
MISCIBLE IN BOTH ALCOHOL (DEHYDRATING
AGENT) AND MELTED PARAFFIN
(IMPREGNATION/INFILTRATION MEDIUM)
• PROCESS OF REMOVING ALCOHOL FROM
SECTION OF TISSUE BY IMMERSING THEM.
• IT REMOVES SUBSTANTIAL AMOUNT OF FAT
FROM TISSUES WHICH OTHERWISE PRESENTS
A BARRIER TO WAX INFILTRATION.
TYPICAL CLEARING SEQUENCE FOR SPECIMENS:
• XYLENE – 20 MINS
• XYLENE – 20 MINS
• XYLENE – 45 MINS
WAX INFILTRATION/IMPREGNATION
• CLEARING AGENT IS COMPLETELY REMOVED
FROM TISSUE AND REPLACED BY A MEDIUM
THAT WILL COMPLETELY FILL ALL TISSUE
CAVITIES
• TISSUE CAN BE INFILTRATED WITH A
SUITABLE HISTOLOGICAL WAX.
• A TYPICAL WAX IS LIQUID AT 60C AND CAN BE
INFILTRATED INTO TISSUE AT THIS
TEMPERATURE THEN ALLOWED TO COOL AT
20C. (E.G. PARAPLAST)
• TISSUE IS PLACED IN A SMALL MOLD. (TISSUE
CASSETTES/CASE/ PLASTIC BOATS)
CONTAINING MELTED PARAFFIN WHICH IS
THEN ALLOWED TO HARDEN.
• PARAFFIN WAX: MOST COMMON; SIMPLEST
SUBSTITUTE: PARAPLAST
TYPICAL INFILTRATION SEQUENCE FOR SPECIMENS:
• WAX – 30 MINS
• WAX – 30 MINS
 • WAS – 45 MINS • INDEX FINGER

EMBEDDING / SECTION CUTTING / STAINING • SPATULA

EMBEDDING • FLAT-BLADED FORCEPS

• SPECIMEN THOROUGHLY INFILTRATED WITH • FISHING OUT:

WAX MUST BE FORMED INTO A “BLOCK”
WHICH CAN BE CLAMPED IN A MICROTOME
FOR SECTION CUTTING.
• PROCESS BY WHICH THE IMPREGNATED
TISSUE IS PALCED INTO PRECISELY ARRANGED
POSITION IN A MOLD CONTAINING MEDIUM
WHICH IS THEN ALLOWED TO SOLIDIFY
• THE WRINKLED SECTIONS ARE
“FLOATED-OUT” IN FLOATATION
WATER BATH FOR 5-10MINS UNTIL
FLATTENED AND THEN “FISH-OUT”
WITH A SLIDE WITH ADHESIVE.
• WATER BATH: 45-50C

TRIMMING • METHODS OF DRYING SLIDE:

• PROCESS OF REMOVING EXCESS WAX FROM • WAX OVEN: 56-60C FOR 2 HRS
THE BLOCK, SO THAT IT FORMS “4-SIDED
• INCUBATOR: 37C OVERNIGHT
PRISM”
• HOT PLATE: 45-55C FOR 45 MINS
• ATLEAST 2MM OF WAX SHOU.D SURROUND
THE TISSUE BLOCK • BLOWER TYPE SLIDE DRYER 50-55C
FOR 20-30 MINS
SECTION CUTTING

• PROCESS WHEREBY TISSUES ARE CUT INTO

UNIFORMLY THIN SLICES OR SECTIONS WITH
THE AID OF A MICROTOME

• PARAFFIN SECTION: 4-6 MICRA

• THE SPECIMEN IS PLACED IN A MICROTOME

FOR SECTIONING AT A THICKNESS DOWN TO
2um TO FORM A RIBBON.

STAINING

• METHOD OF STAINING TISSUES HAVE

THEREFORE BEEN DEVISED THAT NOT ONLY
MAKE VARIOUS TISSUE COMPONENTS
CONSPICUOUS BUT ALSO PERMIT
DISTINCTIONS TO BE MADE BETWEEN THEM.

• TYPES OF DYE/STAIN:

• BASOPHILIC – STAINS NUCLEIC

ACID, GLYCOSAMINOGLYCANS,
AND ACID GLYCOPROTEIN (E.G.
TOLUIDINE BLUE, ALCIAN BLUE,
METHYLENE BLUE, HEMATOXYLIN)

• ACIDOPHILIC – STAINS
MITOCHONDRIA, SECRETORY
GRANULES, AND COLLAGEN (E.G.
ORANGE G, EOSIN, AND ACID
FUCHSIN)

• SECTION CUT IS PICKED BY: