Viability assessment of Ascaris suum eggs stained with fluorescent dyes using digital colorimetric analysis

Abstract

The aim of the study was to develop a method for the colorimetric evaluation of nematode eggs using appropriate instruments. The materials for the study were live and dead (inactivated) eggs of the Ascaris suum. Viability of the eggs was assessed using four different kits for fluorescent staining (for each technique, a series of photos were taken). Images of stained eggs were analysed using graphic software with RGB (red-green-blue) function. The viability of the eggs was assessed according to the relative positions of the distributions of colour intensities of live or dead eggs - distributions area's overlap index (DAOI), and distributions area's separation index (DASI) were calculated. Computer analysis of the intensity of green colour was not satisfactory. However, analysis of images in the spectrum of red colour proved useful for the effective differentiation between live or dead eggs. The best parameters were observed using the Annexin V FITC Apoptosis Detection Kit (DASI = 41 and 67). The investigation confirmed the usefulness of fluorescent dyes used in conjunction with digital analysis for the assessment of the viability of A. suum eggs. The use of computer software allowed a better objectivity of the assessment, especially in the case of doubtful staining.