INTRODUCTION AND MOLECULAR COMPOSITION, PIECES OF

ORGANS SHOULD BE PROMPTLY AND

HISTOLOGY ADEQUATELY TREATED BEFORE, OR AS SOON
• THE STUDY OF THE TISSUES OF THE BODY AS POSSIBLE, AFTER REMOVAL FROM THE
AND HOW THESE TISSUES ARE ARRANGED TO BODY.
CONSTITUTE ORGANS. • PRIMARY AIM: PRESERVE
• FROM THE GREEK WORD HISTO MEANING MORPHOLOGY OF CELL
“TISSUES” OR “WEB”. *WEB BECAUSE OF • SECONDARY AIM: HARDEN TISSUE
INTERWOVEN FILAMENTS AND FIBERS, BOTH
TO PROTECT FROM TRAUMA
CELLULAR AND NONCELLULAR, WITH
MEMBRANOUS LINING. • FORMALDEHYDE AND GLUTARALDEHYDE –
WIDELY USED FIXATIVE.
• IT INVOLVES ALL ASPECTS OF TISSUE
BIOLOGY. *WITH THE FOCUS ON HOW CELLS’ • FORMALDEHYDE: BEST GENERAL
STRUCTURE AND ARRANGEMENT OPTIMIZE FIXATIVE / BEST FOR IRON
FUNCTIONS SPECIFIC TO EACH ORGAN. CONTAINING TISSUES

PREPARATION OF TISSUE FOR STUDY: • FORMALIN – ONE OF THE BEST FIXATIVES

FOR ROUTINE LIGHT MICROSCOPY. *A
*THE MOST COMMON PROCEDURE USED IN
BUFFERED ISOTONIC SOLUTION OF 37%
THE STUDY OF TISSUES IS THE PREPARATION
FORMALDEHYDE.
OF HISTOLOGICAL SECTIONS OR TISSUE
SLICES THAT CAN BE STUDIED WITH THE AID • TISSUES ARE PLACED IN FIXATIVES
OF THE LIGHT MICROSCOPE. ASAP AFTER REMOVAL FROM THE
BODY
BASIC STEPS IN TISSUE PREPARATION FOR
HISTOLOGY: MAIN FACTORS INVOLVED IN FIXATION
• FIXATION • PH: 6-8 OPTIMUM
• DEHYDRATION • TEMPERATURE: GENERALLY, AT RT
• CLEARING • ELECTRON MICROSCOPY &
HISTOCHEMISTRY: 0-4C (TO
• INFILTRATION OR
PREVENT DENATURATION OF
IMPREGNATION
ENZYMES AND PROTEINS)
• EMBEDDING, CASTING OR
• MAST CELLS: RT
MOLDING
• NUCLEIC ACIDS: HIGHER TEMP
• BLOCKING
(RNA: 45C/ DNA: 65C)
• TRIMMING
• THICKNESS OF SECTION: 2-4mm
• SECTIONING OR CUTTING
• CONCENTRATION: 10% FORMALIN
• STAINING
PRACTICAL CONSIDERATIONS:
• MOUNTING
• SPEED: ASAP
LABELING *IMMEDIATELY AFTER REMOVAL OF TISSUE
• PENETRATION: FORMALIN 1mm/hr
FIXATION
• VOLUME: 20X
• THIS METHOD CAN BE DONE BY CHEMICAL
DECALCIFICATION
OR LESS FREQUENTLY PHYSICAL METHODS.
• COMPLETE REMOVAL OF CALCIUM SALTS
• TO AVOID TISSUE DIGESTION BY ENZYMES
FROM THE TISSUE FOLLOWING FIXATION
PRESENT WITH THE CELLS (AUTOLYSIS) OR BY
BACTERIA AND TO PRESERVE THE STRUCTURE
 • RECOMMENDED FLUID TO TISSUE RATIO: • 100 % ETHANOL – 45 MINS
20:1
APPLICATION OF CLEARING:
• IDEAL TIME REQUIRED: 24-48 HRS (2-3 DAYS)
*TO MAKE TISSUE, EMBRYOS, PARASITES
• AT 55C, TISSES WILL UNDERGO COMPLETE TRANSPARENT.
DIGETSION WITHIN 24-48 HRS.
* FOR DEALCOHOLIZATION OF TISSUE
• THE HIGHER THE TEMP, FASTER PREPARATORY TO WAX IMPREGRATION
DECALCIFICATION AND HIGH RISK
OF DAMAGING TISSUE CLEARING

• THE PROCESS WILL DISPLACE THE ETHANOL

TYPES OF DECALCIFYING AGENTS:
IN THE TISSUE. *THE CLEARING SOLUTION IS
• ACID MISCIBLE IN BOTH ALCOHOL (DEHYDRATING
AGENT) AND MELTED PARAFFIN
• NITRIC ACID / HYDROCHLORIC (IMPREGNATION/INFILTRATION MEDIUM)
ACID / FORMIC ACID
• PROCESS OF REMOVING ALCOHOL FROM
• *TISSUE SOFTENERS: FOR UNDULY SECTION OF TISSUE BY IMMERSING THEM.
HARD TISSUES WHICH MAY
DAMAGE THE MICROTOME KNIVES • IT REMOVES SUBSTANTIAL AMOUNT OF FAT
FROM TISSUES WHICH OTHERWISE PRESENTS
• 4% PHENOL / 2%HCL A BARRIER TO WAX INFILTRATION.
DEHYDRATION TYPICAL CLEARING SEQUENCE FOR SPECIMENS:
• MOST OF THE WATER ARE REMOVED • XYLENE – 20 MINS
• PROCESS OF REMOVING EXTRACELLUALR • XYLENE – 20 MINS
AND INTRACELLULAR WATER FROM TISSUE
FOLLOWIUNG FIXATION AND PRIOR TO WAX • XYLENE – 45 MINS
INFILTRATION
WAX INFILTRATION/IMPREGNATION
• THIS IS COMMONLY CARRIED OUT BY
• CLEARING AGENT IS COMPLETELY REMOVED
IMMERSING SPECIMENS IN A SERIES OF
FROM TISSUE AND REPLACED BY A MEDIUM
ETHANOL SOLUTIONS INCREASING THE
THAT WILL COMPLETELY FILL ALL TISSUE
CONCENTRATION. *UNTIL PURE, WATER-
CAVITIES
FREE ALCOHOL IS REACHED. WHICH
EFFECTIVELY REMOVES ALL WATER FROM • TISSUE CAN BE INFILTRATED WITH A
THE TISSUE. SUITABLE HISTOLOGICAL WAX.
• MOST COMMON: ALCOHOL • A TYPICAL WAX IS LIQUID AT 60C AND CAN BE
INFILTRATED INTO TISSUE AT THIS
• ETHANOL: FOR ROUTINE
TEMPERATURE THEN ALLOWED TO COOL AT
DEHYDRATION: EST DEHYDRATING
20C. (E.G. PARAPLAST)
AGENT
• TISSUE IS PLACED IN A SMALL MOLD. (TISSUE
• METHANOL
CASSETTES/CASE/ PLASTIC BOATS)
TYPICAL DEHYDRATION SEQUENCE FOR SPECIMENS: * CONTAINING MELTED PARAFFIN WHICH IS
NOT MORE THAN 4mm THIICK THEN ALLOWED TO HARDEN.

• 70% ETHANOL – 15 MINS • PARAFFIN WAX: MOST COMMON; SIMPLEST

• 90% ETHANOL – 15 MINS SUBSTITUTE: PARAPLAST

• 100 % ETHANOL – 15 MINS TYPICAL INFILTRATION SEQUENCE FOR SPECIMENS:

• 100 % ETHANOL – 15 MINS • WAX – 30 MINS

• 100 % ETHANOL – 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 • THE WRINKLED SECTIONS ARE
FOR SECTION CUTTING. “FLOATED-OUT” IN FLOATATION
WATER BATH FOR 5-10MINS UNTIL
• PROCESS BY WHICH THE IMPREGNATED FLATTENED AND THEN “FISH-OUT”
TISSUE IS PALCED INTO PRECISELY ARRANGED WITH A SLIDE WITH ADHESIVE.
POSITION IN A MOLD CONTAINING MEDIUM
WHICH IS THEN ALLOWED TO SOLIDIFY • 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: