An original simple technique for diagnosis of Microsporidia using glycerol jelly direct smear

Abstract

Microsporidia spp. are obligate intracellular parasites which are very minute with sizes ranging from 1 to 10 μm. They have been increasingly recognized as human pathogens in AIDS and immunocompromised patients, mainly associated with life-threatening chronic diarrhea and systemic disease. For accurate identification of Microsporidia, permanent staining techniques are used to enable the examiner to use the ×100 objective which reveals the important details needed for diagnosis. On the other hand, ×10 and ×40 objectives are of no value in detection of such a minute organism. Until now, there is no study that demonstrates a rapid satisfactory technique for routine examination of wet mount by the oil-immersion lens. Glycerol jelly (GJ) reagent was previously studied for its benefit in fixing the cover slide of direct wet mounts instantly enabling the use of oil-immersion lens in examination that magnifies its role as a rapid technique for direct examination. The aim of this research is to identify Microsporidia by wet mounts immediately, using GJ reagent that enables the examiner to use the ×100 objective and to evaluate GJ wet mount as a method of identification. Glycerol jelly reagent was prepared (7 g gelatin dissolved in 50 ml boiling water was added to 10 ml glycerol) and added to fecal wet mounts stained by iodine and methylene blue. Wet mounts were examined using the ×100 objective. Satisfactory results were achieved in spite of the small size of Microsporidia, as both iodine and methylene blue stained the cytological structures; GJ reagent fixed the cover slide, maintained the high translucency of the films, and enabled the examiner to use the ×100 oil-immersion objective. We also compared fecal wet mounts stained by iodine and methylene blue + GJ with a stool sample stained by permanent stain modified Ziehl-Neelsen without GJ, and we found that fecal wet mounts stained by iodine and methylene blue + GJ were more clear. We concluded that glycerol jelly wet mount is an easy, fast, reliable, and cheap technique for identification of Microsporidia in direct smear, using the ×100 oil-immersion objective.