Evaluation of four fixative-stain combinations for identification of intestinal protozoa in faecal sepcimens: a comparative study

ABSTRACT

Identification of some protozoa in faecal specimens using routine microscopy is sometimes difficult even for the experienced specialist. In order to reach a definite diagnosis, different stains have been routinely used. Most of the routinely used permanent stains require prior preservation of specimens in hazardous fixative. The objective of the present study is to evaluate four fixative-stain combinations for identification of intestinal protozoa in faecal specimens. Fresh stool specimens from 125 patients were divided into aliquots to which each of three different fixatives was added. Specimens were processed and stained with four different stains. A triple stain mixture [Chlorazol Black E, Fast Green and Jenner's stains] was used for specimens preserved in a Cobalt-Zinc based modified PVA fixative [Co-Zn-PVA]. Lacto-phenol cotton blue [LPCB] and modified iron Haematoxylin [MIHX] stains were used for staining of stool fixed in sodium acetate formaldehyde [SAF]. EcoStain was used for staining of EcoFix preserved stool. The first two stains were used as wet mount stains, while the later two were used for preparation of permanently stained slides. Regarding protozoan recovery, the significant figures were that of Entamoeba spp. when identified by LPCB and EcoStain. The stains identified Entamoeba spp. in 12% and 12.8% respectively of stool specimens which were significantly higher than those detected by each of the triple stain mixture and MIHX stains [8%] [p < 0.05]. Regarding the clarity of morphological details, both Triple stain and EcoStain worked better for flagellates, LPCB and EcoStain better identify trophozoites of Entamoeba spp., while both MIHX and triple stain worked better for cysts of Entamoeba spp. The stains which offered easy identification of protozoa from the contrasting background colours were EcoStain and Triple stain mixture. In addition, MIHX stain identified acid fast protozoa along with other intestinal protozoa in the same slide. There are more acceptable alternatives to the traditional methods of fixation and staining. The quality of staining partly depends on a properly matched fixative. According to the multi-attribute ranking of the four used fixative-staining techniques, EcoStain and EcoFix provided the best quality of staining