Module 1 (INTRODUCTION TO HISTOLOGY) Flashcards
THE STUDY OF THE TISSUES OF THE BODY AND HOW THESE TISSUES ARE ARRANGED TO CONSTITUTE ORGANS.
HISTOLOGY
FROM THE GREEK WORD HISTO MEANING “TISSUES” OR “WEB”. *WEB BECAUSE OF INTERWOVEN FILAMENTS AND FIBERS, BOTH CELLULAR AND NONCELLULAR, WITH MEMBRANOUS LINING.
HISTOLOGY
IT INVOLVES ALL ASPECTS OF TISSUE BIOLOGY. *WITH THE FOCUS ON HOW CELLS’ STRUCTURE AND ARRANGEMENT OPTIMIZE FUNCTIONS SPECIFIC TO EACH ORGAN.
HISTOLOGY
GREEK WORD HISTO MEANING
“TISSUES” OR “WEB
THIS METHOD CAN BE DONE BY CHEMICAL OR LESS FREQUENTLY PHYSICAL METHODS.
FIXATION
TO AVOID TISSUE DIGESTION BY ENZYMES PRESENT WITH THE CELLS (AUTOLYSIS) OR BY BACTERIA AND TO PRESERVE THE STRUCTURE AND MOLECULAR COMPOSITION, PIECES OF ORGANS SHOULD BE PROMPTLY AND ADEQUATELY TREATED BEFORE, OR AS SOON AS POSSIBLE, AFTER REMOVAL FROM THE BODY.
FIXATION
PRESERVE MORPHOLOGY OF CELL
FIXATION primary aim
HARDEN TISSUE TO PROTECT FROM TRAUMA
FIXATION secondary aim
WIDELY USED FIXATIVE
FORMALDEHYDE AND GLUTARALDEHYDE
BEST GENERAL FIXATIVE / BEST FOR IRON CONTAINING TISSUES
FORMALDEHYDE
ONE OF THE BEST FIXATIVES FOR ROUTINE LIGHT MICROSCOPY. *A BUFFERED ISOTONIC SOLUTION OF 37% FORMALDEHYDE.
FORMALIN
MAIN FACTORS INVOLVED IN FIXATION
pH
Temperature
Thickness of Section
Concentration
pH factor in fixation
6-8 OPTIMUM
temperature factor in fixation
GENERALLY AT RT
thickness of section factor in fixation
2-4mm
concentration factor in fixation
10% Formalin
Practical considerations in fixation
Speed: ASAP
Penetration: Formalin 1mm/hr
Volume: 20x
COMPLETE REMOVAL OF CALCIUM SALTS FROM THE TISSUE FOLLOWING FIXATION
DECALCIFICATION
RECOMMENDED FLUID TO TISSUE RATIO in Decalcification
20:1
IDEAL TIME REQUIRED of Decalcification
24-48 HRS (2-3 DAYS)
TISSES WILL UNDERGO COMPLETE DIGETSION within (___) and at what temperature.
24-48 HRS at 55 C
Types of Decalcifying agents
Acid: Nitric acid/ hydrochloric acid/ formic acid
Tissue Softeners: 4% phenol/ 2% HCL
Most of the water are removed
Dehydration
Process of removing extracellular and intracellular water from tissue following fixation and prior to wax infiltration
Dehydration
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
Dehydration
MOST COMMON used dehydrating agent
Alcohol
Alcohol used in dehydration
Ethanol : for routing dehydration : EST dehydrating agent
Methanol
TYPICAL DEHYDRATION SEQUENCE FOR SPECIMENS
not more than 4mm THICK
TYPICAL DEHYDRATION SEQUENCE FOR SPECIMENS:* NOT MORE THAN 4mm THICK
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 IMPREGNATION
THE PROCESS WILL DISPLACE THE ETHANOL IN THE TISSUE
CLEARING
SOLUTION IS MISCIBLE IN BOTH ALCOHOL (DEHYDRATING AGENT) AND MELTED PARAFFIN (IMPREGNATION/INFILTRATION MEDIUM)
CLEARING
PROCESS OF REMOVING ALCOHOL FROM SECTION OF TISSUE BY IMMERSING THEM
CLEARING
IT REMOVES SUBSTANTIAL AMOUNT OF FAT FROM TISSUES WHICH OTHERWISE PRESENTS A BARRIER TO WAX INFILTRATION.
CLEARING
TYPICAL CLEARING SEQUENCE FOR SPECIMENS
XYLENE – 20 MINS
XYLENE – 20 MINS
XYLENE – 45 MINS
CLEARING AGENT IS COMPLETELY REMOVED FROM TISSUE AND REPLACED BY A MEDIUM THAT WILL COMPLETELY FILL ALL TISSUE CAVITIES
WAX INFILTRATION/IMPREGNATION
TISSUE CAN BE INFILTRATED WITH A SUITABLE HISTOLOGICAL WAX
WAX INFILTRATION/IMPREGNATION
CAN BE INFILTRATED INTO TISSUE AT THIS TEMPERATURE THEN ALLOWED TO COOL AT 20C. (E.G. PARAPLAST)
WAX INFILTRATION/IMPREGNATION
A TYPICAL WAX IS
LIQUID AT 60C
Tissue is placed in a small mold. (Tissue cassettes/ case/ plastic boats) containing melted paraffin which is then allowed to harden.
WAX INFILTRATION/IMPREGNATION
MOST COMMON ; SIMPLEST reagent in Wax Infiltration/Impregnation
Paraffin Wax
Substitute of Paraffin wax
paraplast
TYPICAL INFILTRATION SEQUENCE FOR SPECIMENS
WAX – 30 MINS
WAX – 30 MINS
WAS – 45 MINS
SPECIMEN THOROUGHLY INFILTRATED WITH WAX MUST BE FORMED INTO A “BLOCK” WHICH CAN BE CLAMPED IN A MICROTOME FOR SECTION CUTTING.
EMBEDDING
PROCESS BY WHICH THE IMPREGNATED TISSUE IS PALCED INTO PRECISELY ARRANGED POSITION IN A MOLD CONTAINING MEDIUM WHICH IS THEN ALLOWED TO SOLIDIF
EMBEDDING
PROCESS OF REMOVING EXCESS WAX FROM THE BLOCK,SO THAT IT FORMS “4-SIDED PRISM
TRIMMING
ATLEAST 2MM OF WAX SHOULD SURROUND THE TISSUE BLOCK
TRIMMING
PROCESS WHEREBY TISSUES ARE CUT INTO UNIFORMLY THIN SLICES OR SECTIONS WITH THE AID OF A MICROTOME
SECTION CUTTING
PARAFFIN SECTION : 4-6 MICRA
SECTION CUTTING
THE SPECIMEN IS PLACED IN A MICROTOME FOR SECTIONING AT A THICKNESS DOWN TO 2um TO FORM A RIBBON.
SECTION CUTTING
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.
STAINING
TYPES OF DYE/STAIN:
BASOPHILIC
ACIDOPHILIC
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
INDEX FINGER
SPATULA
FLAT-BLADED FORCEPS
THE WRINKLED SECTIONS ARE “FLOATED-OUT” IN FLOATATION WATER BATH FOR 5-10MINS UNTIL FLATTENED AND THEN “FISH-OUT” WITH A SLIDE WITH ADHESIVE.
FISHING OUT
WATER BATH temperature for fishing out
45-50 C
METHODS OF DRYING SLIDE
WAX OVEN : 56-60C FOR 2 HRS
INCUBATOR : 37C OVERNIGHT
HOT PLATE : 45-55C FOR 45 MINS
BLOWER TYPE SLIDE DRYER 50-55C FOR 20-30 MINS
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
