An investigation of the fixation and staining of lipids by a combination of malachite green or other triphenylmethane dyes with glutaraldehyde

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Abstract
Mixtures of the monocationic triphenylmethane dyes, malachite green or crystal violet, with glutaraldehyde, retained and stained phospholipid droplets in chloroplasts of leaves of Lolium multiflorum Lam. These dyes also stained trilinolenin; phosphatidic acid dioleoyl, dipalmitoyl; phosphatidylcholine dioleoyl, dilinoleoyl; and phosphatidylethanolamine dioleoyl on filter paper models. In this model system the dipalmitoyl derivatives of phosphatidylcholine and phosphatidylethanolamine did not stain well, if at all. Washing of the dyed samples with 0.5 M sodium chloride solution did not remove the colour, suggesting that the interaction is unlikely to be purely ionic. Except with trilinolenin, the colour and possibly the lipid samples were removed from the filter paper model system on washing with 100% ethyl alcohol. Other triphenylmethane dyes (methyl green, light green and fast green FCF) did not retain phospholipid droplets in tissue. Fast green did, however, stain phospholipids in the model system. Two quinone-imine dyes, neutral red and toluidine blue O, while staining phospholipids in the model system did not retain droplets on the chloroplasts but did assist in the retention and staining of cell membranes. The basis of the reaction between lipid and dye is discussed in relation to the structural formulae of the dyes and model lipids. It is possible that there is an interaction between the hydrophobic fatty acid ester side chains of the lipid and the dyes. Neither the phosphate nor the polyhydric alcohol moieties of the lipid seem to be essential for staining or retention of lipid.