Fixation Part 5

● Pale yellow powder in water (6% in 20ºC)

● Ultrathin section in Electron Microscopy

OSMIC ACID FIXATIVES

DADV: very expensive, very volatile, inhibits hematoxylin

OSMIC ACID FIXATIVES

Tissue-to-fixative ratio: 1:5

OSMIC ACID FIXATIVES

Nuclear fixative (24 - 48hrs)

Flemming’s Solution w/ GAC

Most common osmic acid fixative

Flemming’s Solution w/ GAC

Effect: permanently fixes fat

Flemming’s Solution w/ GAC

ADV: needs less amount of fixative

Flemming’s Solution w/ GAC

Cytoplasmic Fixative (24 - 48hrs)

Flemming’s Solution w/o GAC

○ Incorporated into compound fixatives

Trichloroacetic Acid

○ Marked swelling effect on tissues

Trichloroacetic Acid

○ Poor penetration thus for small pieces of tissues or bones

Trichloroacetic Acid

○ Weak decalcifying agent, this has softening effect on dense fibrous tissues

Trichloroacetic Acid

○ Use at ice cold temperature (-5 to 4ºC)

Acetone

○ For water-diffusible enzymes (Phosphatase, Lipase)

Acetone

○ Fixes brain tissue (for rabies)

Acetone

DADV: Dissolves fat, evaporates rapidly

Acetone

Involves thermal coagulation of tissue proteins for rapid diagnosis

HEAT FIXATION

Frozen tissue sections & bacteriologic smears

HEAT FIXATION

10 - 15mins thickness of tissue

HEAT FIXATION

Increases movement of molecules to accelerate fixation, staining, and decalcification

Microwave Technique (Heat Fixation)

ADV: Can preserve neurochemical substances of tissues

DADV: Cannot penetrate tissues that are 10 - 15mm in thickness

HEAT FIXATION

Optimum temperature of HEAT FIXATION

45º - 55ºC

● To improve the demonstration of particular substances

● To make special staining possible (fixatives as mordants)

● To ensure further and complete hardening and preservation of tissues

SECONDARY FIXATION

○ Technique whereby a primary fixed tissue is placed in aq. Solution of 2.5% - 3% potassium dichromate for 24hrs

Post-Chromatization

Process of removing excess fixatives

● Tap water → Excess chromates, formalin, osmic acid

● 50% - 70% Alcohol → Picric acid

● Alcoholic Iodine → Mercuric fixatives

WASHING OUT

DIFFICULTIES IN IMPROPER FIXATION

● Failure to arrest autolysis

● Tissues are brittle and too hard → Too long fixation time

● Shrinkage and swelling of cells → No balance

● Tissue soft consistency

● Presence of artifact pigments

● Removal of soluble substances

● Incomplete result in other procedures