Huh
Huh
Huh
School of Health Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia,
16150 Kubang Kerian, Kelantan, Malaysia
3 Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800, Penang, Malaysia.
4 Laboratory Animal Research Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.
5 Faculty of Applied Sciences, AIMST University, 08000 Sungai Petani, Kedah, Malaysia.
6 Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autnoma de Mxico,
04510 Mxico D.F., Mxico
Corresponding author e-mail: limbh493@gmail.com
2
Abstract. Entamoeba histolytica causes about 50 million infections worldwide with a death rate
of over 100,000 annually. In endemic developing countries where resources are limited, microscopic
examinations based on Wheatley trichrome staining is commonly used for diagnosis of intestinal
amoebiasis. Other than being a time-consuming method, it must be performed promptly after
stool collection as trophozoites disintegrate rapidly in faeces. The aim of this study was to compare
the efficacies of Eosin-Y, Wheatley trichrome and Iodine stains in delineating the diagnostic features
of the parasite, and subsequently to determine the suitable microscopy observation period for
detection of erythrophagocytic and non-erythrophagocytic trophozoites spiked in semi-solid stool
sample. Wheatley trichrome staining technique was performed using the standard method while
the other two techniques were performed on the slides by mixing the respective staining solution
with the spiked stool sample. One million of axenically cultured non-erythrophagocytic E.
histolytica and erythrophagocytic E. histolytica were separately spiked into 2 g of fresh semisolid faeces. Percentage viability of the trophozoites in the spiked stool sample was determined
at 30 minute intervals for eight hours using the 0.4% Trypan blue exclusion method. The results
showed that Eosin-Y and Wheatley trichrome stained the karyosome and chromatin granules
better as compared to Iodine stain. The percentage viability of non-erythrophagocytic trophozoites
decreased faster than the erythrophagocytic form in the first 5 hours and both dropped to ~10% in
the 6th hour spiked sample. In conclusion, Eosin-Y staining technique was found to be the easiest
to perform, most rapid and as accurate as the commonly used Wheatley trichrome technique;
Eosin-Y stained slide sealed with DPX could also be kept as a permanent record. A period not
exceeding 6 hours after stool collection was found to be the most suitable in order to obtain good
microscopy results of viable trophozoites.
INTRODUCTION
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Figure 1. Stained trophozoites. (a) Wheatley trichrome stained trophozoite, 1000X magnification (b)
Iodine stained trophozoite, 1000X magnification (c) Eosin-Y stained trophozoite, 1000X magnification
(d) Eosin-Y stained erythrophagocytic trophozoite, 1000X magnification (e) Eosin-Y stained trophozoite
showed clear chromatin granules and karyosome, 400X magnification (f) Eosin-Y stained trophozoite
without DPX seal indicated unclear nuclear characteristics after an hour, 400X magnification.
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