A double staining method using calcofluor white and acridine orange to differentiate life stages of Enterocytozoon hepatopenaei (EHP) on hepatopancreatic sections

A double fluorescent staining method was used to differentiate life stages of Enterocytozoon hepatopenaei (EHP) with a chitin-binding dye, calcofluor white (CFW) and a nucleic acid dye, acridine orange (AO). The unreleased spores showed a significantly blue fluorescent chitin wall with weak orange-red RNA staining, while the plasmodia appeared grape cluster-like orange-red nucleic acid-containing particles without or with very weak blue fluorescence and sporophytes showed grape cluster-like orange-red particles with varying degree of blue fluorescence. The analysis of the red, blue and green intensity of different targets revealed that the ratio of relative concentration of CFW and AO (RRC/A) of plasmodia (0.36 ± 0.15) (n = 60), sporophytes (1.25 ± 0.41) (n = 157), and unreleased spores (3.06 ± 1.09) (n = 21) had extremely significant differences (P