Histopathologic Techniques: Fixatives, Chapter 3 Part III Formaldehyde (Gregorios)

10% formalin

most widely used fixative

40% formalin

pures stock solution of formalin

Formalin fixation time

24 hours

Formalin if unbuffered

reduces basophilic and eosinophilic staining of cells; it forms abundant brown pigment granules on blood-containing tissues e.g., spleen, due to blackening of hgb

Prolonged fixation with formalin

bleaching of specimen and loss of natural colors; dispersal of fat from the tissues into the fluid; dissolution or loss of glycogen, biurate of sodium crystals and uric acid

Paraformaldehye deposits

caused by prolonged storage of formaldehyde at very low temperature

precipitates may be removed by filtration or by adding of 10% methanol

Concentrated formaldehyde

must NEVER be neutralized since this might cause explosion

Brown or black crystalline precipitate

formed by the action of formic acid with blood

this can be removed by treatment with saturated alcoholic picric acid or a 1% solution of potassium hydroxide in 80% alcohol

neutral phosphate buffered formalin (prevent pigmentation)

Fatty tissues that are to be stored for a long time

cadmium and cobalt salts are added to prevent dispersion of fat out into the fluid

Acid reaction due to formic acid formation

this can be buffered/neutralized by adding magnesium carbonate or calcium carbonate

To improve staining and produce firmer and harder consistency

tissues fixed in formalin for 1-2 hours
place in Helly's fluid for 4-6 hours or in formol-sublimate for 4-16 hours (secondary fixation)

If postfixed in osmic acid

tissue must not be washed in demineralized water to prevent hypotonicity and bleaching

Blocks not exceeding in 5mm in thickness

complete in 6-12 hours at room temperature

Heat
Vacuum
Agitation
Microwave

techniques that could influence fixation by formalin

50°C

fixation can be speeded up by heating at this temperature and by placing it under a vacuum or in a microwave