Schiff reactions with lipids and the disputed terminal rinse with hydrochloric acid

Summary

We have been concerned with the effect of 3N HCl on the reaction-products of lipids and Schiff's reagent in the PAS and plasmal reactions. Our conclusions are:

• 1. We agree with Elleder and Lojda (1971) that the intensity of the modified PAS and plasmal reactions is reduced if a terminal 3N HCl rinse is used. However, we consider that the term “rinse” is not synonomous with a 5–10 min wash or bath.

• 2. We do not agree that this reduction is substantial. After a 1 min rinse with 3N HCl, it amounts to only 9.1% with the plasmal and 25.4% with the modified PAS reaction.

• 3. We do not agree that this modest reduction is necessarily all due to the release of fatty (or glycollic) aldehydes from conjugation with Schiff's reagent; it could equally well be due to suppression of phospholipid (or other anion) staining by the residual basic fuchsin in Schiff's reagent.

• 4. We do not agree that the plasmal (or PAS) reaction of brain lipids on paper is nearly extinguished by a terminal rinse in 3N HCl: such a result is possibly due to imperfect washing of chromatograms. With a terminal rinse of 1 min, the reactions were reduced by 31.5% and nil, respectively.

• 5. We do not agree that “the influence of an acid mounting medium” is of any practical significance with these histochemical reactions.

• 6. Finally we wish to reiterate that the staining of phospholipids (or other anions) by the residual basic fuchsin in Schiff's reagent is potentially a very real hazard in the application of the modified PAS and plasmal methods. Our experiments suggest that this hazard can be obviated by the use of the brief terminal 3N HCl rinse. Even if the acid does remove part of the reaction product with some Schiff methods, it is perhaps safer on these occasions to lose some of the baby with the bath-water.