Plaque Formation of Toxoplasma gondii in Vero Cells using Carboxymethylcellulose

— Toxoplasma gondii is an intracellular parasite capable of infecting all nucleated cells in a diverse array of species. Toxoplasma plaque assay have been described using Bacto Agar. Because of its experimental advantages carboxymethyl cellulose overlay, medium viscosity was choosing and the aim of this work was to develop alternative method for formation of T. gondii plaques. Tachyzoites were inoculated onto monolayers of Vero cells and cultured at 37° C under 5 % CO 2 . The cultures were followed up by microscopy inspection. Small plaques were visible by naphtol blue stain 4 days after infection. Larger plaques could be observed by day 10 of culture. The carboxymethyl cellulose is a cheap reagent and the methodology is easier, faster than assays under agar overlay. This is the first description of the carboxymethyl cellulose overlay use for obtaining the formation of T. gondii plaques and may be useful in consequent obtaining tachyzoites for detailed studies.

[1]  Yongxin Hao,et al.  Expression of the tandem enhanced yellow fluorescent marker gene in Toxoplasma gondii , 2009, Parasitology Research.

[2]  W. Rivera,et al.  Colony growth of Philippine isolates of Blastocystis hominis in simplified, soft agar medium , 2007, Parasitology Research.

[3]  H. Klenk,et al.  New low-viscosity overlay medium for viral plaque assays , 2006, Virology Journal.

[4]  M. Hess,et al.  Clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and a Blastocystis sp. established through micromanipulation , 2006, Parasitology.

[5]  C. Sreekumar,et al.  In vitro generation of cloned populations of Toxoplasma gondii , 2003, Parasitology Research.

[6]  A. Joss,et al.  Toxoplasma gondii in vitro culture for experimentation. , 2002, Journal of microbiological methods.

[7]  D. H. Hollander Colonial morphology of Trichomonas vaginalis in Agar. , 1976, The Journal of parasitology.

[8]  E. Pfefferkorn,et al.  Toxoplasma gondii: isolation and preliminary characterization of temperature-sensitive mutants. , 1976, Experimental parasitology.

[9]  S. Halstead,et al.  Antibody response in dengue and dengue hemorrhagic fever. , 1967, Japanese journal of medical science & biology.

[10]  S. Chaparas,et al.  Plaque Assay of Toxoplasma on Monolayers of Chick Embryo Fibroblasts.† , 1959, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[11]  M. Müller,et al.  Plaque assay for human coronavirus NL63 using human colon carcinoma cells , 2008, Virology journal.

[12]  D. E. Mueller,et al.  Clonal growth in Petri dishes of Entamoeba histolytica. , 1995, Transactions of the Royal Society of Tropical Medicine and Hygiene.

[13]  D. Roos,et al.  Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. , 1994, Methods in cell biology.

[14]  S. A. Edgar,et al.  A NEW COCCIDIUM OF CHICKENS, EIMERIA MIVATI SP. N. (PROTOZOA: EIMERIIDAE) WITH DETAILS OF ITS LIFE HISTORY. , 1964, The Journal of parasitology.