Difference in the Quantification of Toxoplasma Gondii by Yellow Fluorescent Protein and the Trypan Blue Exclusion Test

Abstract

Purpose: : Toxoplasma gondii is a protozoan parasite that infects up to a third of the world's population. Toxoplasmic infection by bradyzoites and tachyzoites can be either congentinally or postnatally aquired and chorioretinitis can occur as a result of acute infection or latent reactivation. Molecular biology assays, including PCR, are critical in the dignosis of the congenital infection. Laboratory diagnosis of the latent reactivation is inadequate. To validate these technologies, it is important to determine the concentration of T. gondii in a cell suspension, for which the trypan blue exclusion test is used. However, professional training is required for an accurate count. Yellow fluorescent protein (YFP)-expressing T. gondii allows for the visualization of parasites. We sought to investigate the reliablity of both methods in determining the concentration of T. gondii.

Methods: : YFP-expressing RH strain T gondii tachyzoites were grown in monolayers of human foreskin fibroblasts. Cells were cultured in DMEM and tachyzoites were harvested. Both the trypan blue exclusion test and visualization of fluorescence were preformed using a hemacytometer with Neubauer rulings. An inverted fluorescent microscope was used to observe T. gondii at 400x and 200x magnification. Individual samples were counted using both the typan blue exclusion test and fluorescence microscopy.

Results: : Tachyzoites were harvested when approximately 80% of the fibroblast monolayer was lysed. Counting by the trypan blue exclusion test yeilded an average of 2.9 million parasites per millilitre in cell suspension where as, counting by fluoresce yeilded an average of 6.18 million parasites per millilitre in the same sample. The fluorescence microscopy revealed more tachyzoites than the trypan blue exclusion method; the exclusion test underestimated the concentration of the cell suspension by 2.13 times.

Conclusions: : Counting by fluorescent microscopy is a more accurate method of determining the number of T. gondii in a cell suspension than the trypan blue exclusion test. Fluorescent microscopy is user-friendly and requires no special training, only the identification of fluorescent structures. In contrast, the trypan blue exclusion test requires an intimate knowledge of the parasite's morphology. The use of fluorescent microscopy is a valuable tool for identification of T. gondii in a research setting.

Keywords: toxoplasmosis • microscopy: light/fluorescence/immunohistochemistry