Pol Andre Bouin came up with this fixative by the end of XIX century. Since then, it has been widely used for paraffin embedding samples and common staining techniques, for example in histopathology. It is a good choice for soft tissues and embryo samples. The nuclei and glycogen inclusions are well-preserved. In addition, it works as mordant for some dyes. The fixation is carried out by picric acid, that precipitates molecules, and formaldehyde, that cross-links those molecules.
Procedure
15 ml of picric acid saturated solution
5 ml of 40 % formaldehyde
1ml of glacial acetic acid
Picric acid saturated solution: add abundant solid picric acid to distilled H2O. After 2 or 3 days, the H2O is saturated of dissolved picric acid, while precipitate remains at the bottom of the bottle. The saturated H2O can be used to prepare the fixative. More distilled H2O can be added to the bottle to replace the used volume. This can be done as long as solid picric acid is observed at the bottom. If all picric acid is dissolved, more solid picric acid has to be added.
The picric acid saturated solution can be stored for months.
Formaldehyde: 37-40 % formaldehyde in distilled H2O .
Notes
The Bouin fixative can be stored for a long time, as long as the container is tightly closed.
The fixation time has to be experimentally set. However, more than 48 are not recommended for fixations by immersion. After fixation, samples can be stored in 70º ethanol. Bouin fixative is not recommended in kidney and mitochondria studies.
Before paraffin embedding, it is a good practice to remove as much picric acid as possible (the yellow color). This can be done by long washes in 70º ethanol.
See the safety data sheet for the picric acid because it can be explosive under some circumstances.
Products
Formaldehyde
Picric acid
Ácetic acid
Distilled H2O
Labware
Test tube
Bottles
Beakers