FIXATION 1

FIXATION

Definition:

• Classically defined as the killing, penetration or hardening of tissues.
• Currently defined as the alteration of tissues by stabilizing protein so that the tissues become resistant to further changes.

PRIMARY GOAL:

• To preserve the morphology and chemical integrity of the tissue as close to the original as possible.

SECONDARY GOAL:

• To harden the tissue for easier cutting.
• To protect the tissue from trauma of further handling.

FUNCTIONS OF FIXATIVE:

• It changes the soluble contents of cells into insoluble structures that can otherwise be lost during subsequent processing.
• It stops AUTOLYSIS & PUTREFACTION.
• It stabilizes structures to maintain the proper relationship of cells and their stroma.
• It renders the tissues firmer for proper grossing and easy cutting of thin sections for processing.

TYPES OF FIXATIVES:

1. ADDITIVE FIXATIVES

• The chemical component of the fixative becomes part of the tissue.
  EXAMPLES:
• FORMALDEHYDE
• MERCURIC CHLORIDE
• CHROMIUM TRIOXIDE
• PICRIC ACID
• GLUTARALDEHYDE
• OSMIUM TETROXIDE
• ZINC SULFATE / CHLORIDE

2. NON-ADDITIVE FIXATIVES

• The chemical component of the fixative DOES NOT become part of the tissue but ALTERS the tissue component.
  EXAMPLES:
• ALCOHOL FIXATIVES
• ACETONE

FACTORS INVOLVED IN FIXATION:

• HYDROGEN ION CONCENTRATION
• TEMPERATURE
• THICKNESS OF SECTIONS / SPECIMEN SIZE
• OSMOLALITY
• CONCENTRATION
• DURATION OF FIXATION
• PENETRATION

CONSEQUENCES OF DELAYED, INCOMPLETE OR POOR FIXATION:

• Loss or total disappearance of NUCLEAR CHROMATIN.
• Disappearance of some cells.
• CELL SHRINKAGE with artifactual space around the cells.

AMOUNT OF FIXATIVE:

• Should be 20x the volume of the specimen (15–20:1 ratio)

1. ELECTRON MICROSCOPY: 20x the volume of the specimen
2. OSMIUM TETROXIDE: 5–10x the volume of the specimen
3. MUSEUM PREPARATIONS: Not less than 50x the volume of the specimen

FACTORS TO CONSIDER IN CHOOSING THE APPROPRIATE FIXATIVE:

• The need for immediate examination
• The type of tissue to be processed
• The tissue structure being studied
• The type of stain to be used

EFFECTS OF FIXATIVE, IN GENERAL:

• Harden the tissue
• Prevent bacterial growth
• Reduce the risk of infection
• Increase optical differentiation of cells and tissue

FACTORS THAT WILL RETARD / PROLONGED FIXATION TIME:

• SIZE & THICKNESS: THE LARGER, THE LONGER TIME
• COLD TEMPERATURE
• PRESENCE OF MUCUS & BLOOD → WASH WITH NORMAL SALINE
  NOTE: If the specimen is HUMAN BRAIN, wash with RINGER’S LACTATE

FACTORS THAT WILL ACCELERATE FIXATION TIME:

• SIZE & THICKNESS: THE SMALLER, THE SHORTER TIME
• AGITATION: CONTINUOUS MIXING
• HEAT & PRESSURE: 37–56°C

SIMPLE FIXATIVE

• Contains only ONE SUBSTANCE

COMPOUND FIXATIVE

• Contains MORE THAN ONE SUBSTANCE

HEAT FIXATION

• Thermal coagulation of proteins, usually used for BACTERIAL SMEARS & FROZEN SECTIONS.

MICROWAVE TECHNIQUE

• Accelerates FIXATION, DECALCIFICATION & STAINING
• Physical agent with similar mechanism to oven, vacuum, and agitation.
• Maybe used for neurochemical substances like ACETYLCHOLINE.

SECONDARY FIXATION

• Placing an already FIXED TISSUE in a SECOND FIXATIVE in order to:
  • Facilitate and improve DEMONSTRATION OF PARTICULAR SUBSTANCES
  • Make SPECIAL STAINING TECHNIQUES POSSIBLE
  • Ensure further and COMPLETE HARDENING & FORMALIN preservation of tissues.

POST-CHROMATIZATION

• Form of secondary fixation using any CHROMATE FIXATIVE.

WASHING OUT

• Removing EXCESS FIXATIVES.

1. TAP WATER

• Removes excess OSMIC ACID, CHROMATES
  • KELLY’S
  • ZENKER’S
  • FLEMMING’S SOLUTION

2. 50–70% ALCOHOL

• Wash out excess PICRIC ACID

3. ALCOHOLIC IODINE

• Removes excess MERCURIC FIXATIVE