Improved contrast of Acid Fuchsin stain using UV fluorescence microscopy

Abstract

Acid Fuchsin (AF) stain is used for the detection of plant pathogenic nematodes within host roots under light microscopy. Nematodes within host roots appear magenta in colour due to the stain penetrating the impermeable cuticle during the heat treatment step in the staining protocol i.e. boiling in AF solution. AF stained nematode infected plant root samples are stored for a short period in acidified glycerol solution or observed as soon as possible under the microscope. However, if stored for a longer period in acidified glycerol solution, the stain within the nematodes decreases in intensity leading to a loss of contrast between the nematodes and plant roots. AF is a red fluorochrome (Emmax 630nm) under ‘green light’ excitation when UV fluorescence is used. If the microscope is not equipped with a ‘green light’ excitation filter, AF can be excited under ‘blue light’ exhibiting a dull red fluorescence signal at pH values below 6. UV fluorescence under ‘blue light’ excitation was used as an alternative means to observe AF destained Meloidogyne sp. infected tomato root samples stored for a long period in acidified glycerol solution.