Potentially pathogenic free-living amoebae (FLA) are unique protozoa that present a healthcare challenge. The appropriate choice of staining technique is crucial to improving the efficiency of microscopic diagnosis of FLA isolated from environmental as well as clinical samples. This study aimed to evaluate different staining techniques for morphological
identification of Acanthamoeba and Naegleria isolated from tap water samples in different localities in Assiut City, Egypt. A multi-attribute evaluation was performed to compare these techniques considering staining quality, ease of performance, cost and time of each procedure. Seven staining techniques were used including Lugol's iodine and lactophenol cotton blue as temporary stains, with Giemsa, trichrome, iron hematoxylin, acid-fast and Gram stains as permanent stains. Both lactophenol cotton blue and Lugol's iodine showed good structural details of different stages of FLA and good contrast at a very short time and reasonable cost, followed by Giemsa. Both trichrome and iron hematoxylin stains gave comparable staining quality but at a longer time with more complex staining procedures and higher cost. Both modified acid-fast and Gram stains didn't provide sufficient staining quality to identify morphological details of FLA. According to the multi-attribute evaluation, both Lugol's iodine and lactophenol cotton blue achieved the highest rank (92.85%) followed by Giemsa and Gram stains (64.28 %), modified acid-fast stain (57.14%), while iron hematoxylin and Masson's
trichrome had the last rank (46.42%).