Fixation Part 2

Types of Simple Fixatives

I. Aldehyde

II. Metallic Fixatives

III. Picric acid

IV. Glacial acetic acid

V. Alcohol

VI. Osmium Tetroxide

VII. Trichloroacetic acid

VIII. Acetone

IX. Heat

What are the Microanatomical fixatives?

- 10% NBF

- 10% Formol-Saline

- Heidenhain’s Susa

- Zenker’s

- Zenker-formol (Helly’s)

- Bouin’s

- Brail’s

What are the Nuclear fixatives?

- Flemming’s with glacial acetic acid

- Carnoy’s

- Bouin’s

- Newcomer’s

- Heidenhain’s

What are the Cytoplasmic fixatives?

- Helly’s

- Orith’s

- Regaud’s / Molter’s

- Formalin with Post-charming

- Fleming’s w/o glacial acetic acid

What are the Histochemical fixatives?

- 10% Formol Saline

- Absolute ethanol

- Newcomer’s

- Acetone

Gas produced by oxidation of methanol

Formaldehyde (Formalin)

What is the concentration of formalin when it’s in:

■ gas form

100% formalin

What is the concentration of formalin when it’s in:

■ stock concentration (causes overhardening of the external surfaces of tissues)

37% to 40% formalin

What is the concentration of formalin when it’s in:

■ working solution; most commonly used

10% formalin

is Usually buffered to pH 7 with phosphate buffer

Formaldehyde (Formalin)

ADVANTAGES of Formalin

DISADVANTAGES of Formalin

What is the cause of:
White paraformaldehyde precipitates

Prolonged storage

What is the cause of:
Formation of Formic acid

Unbuffered

What is the cause of:
Formalin pigments brown / black precipitate

Action of formic acid with excess blood

What is the Remedy of:
White paraformaldehyde precipitate

- Filter

- Add 10% methanol (but dentures proteins thus unsuitable for EM)

What is the Remedy of:
Formation of Formic acid

- Buffer or Methanol

- 10% formol saline + Mg++ / Ca++ carbonate in jar with marbles

What is the Remedy of:
Formalin pigments brown / black precipitate

- Saturated alcoholic picric acid

- 1% KOH in 80% ROH

- Kardasewitsch’s Method (70% ETOH & 28% ammonia H20)

- Lillie’s MTD (Acetone, H2O2 70% ETOH & 28% ammonia water)

Saturated formaldehyde + 10% NaCl

10% FORMOL-SALINE

recommended for fixation of CNS tissues and general post mortem tissues for histochemical examination.

10% FORMOL-SALINE

ADV: ideal for Silver impregnation staining technique

DADV: slower; tissue shrinks during alcohol dehydration [Remedy: Secondary fixation]

10% FORMOL-SALINE

○ Formaldehyde + Na Dihydrogen Phosphate + Disodium Hydrogen Phosphate

○ Has an ideal pH of 7

10% NEUTRAL BUFFERED FORMALIN

Best general tissue fixative

10% NEUTRAL BUFFERED FORMALIN

Best for iron-containing pigments and elastic fibers which do not stain well after Susa, Zenker or Chromate fixation,

10% NEUTRAL BUFFERED FORMALIN

DADV: longer to prepare, inert to phospholipids and neutral fats

10% NEUTRAL BUFFERED FORMALIN

Fixation Time of 10% NEUTRAL BUFFERED FORMALIN

4 - 24hrs

○ Saturated aq. Mercuric chloride + 40% Formaldehyde

Formol-Corrosive (Formol Mercuric Chloride)

Recommended for routine post mortem tissues and Silver Reticulum staining methods

Formol-Corrosive (Formol Mercuric Chloride)

ADV: does not need washing, fixes lipids

DADV: Forms mercuric chloride deposits

Formol-Corrosive (Formol Mercuric Chloride)