N P Ramachandran

National University of Singapore, Singapore, Singapore

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Publications (4)10.71 Total impact

  • K S Tan, G C Ng, E Quek, J Howe, N P Ramachandran, E H Yap, M Singh
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    ABSTRACT: Colony growth of protozoan parasites in agar can be useful for axenization, cloning, and viability studies. This is usually achieved with the pour plate method, for which the parasite colonies are situated within the agar. This technique has been described for Giardia intestinalis, Trichomonas vaginalis, and Entamoeba and Blastocystis species. Extracting such colonies can be laborious. It would be especially useful if parasites could be grown on agar as colonies. These colonies, being exposed on the agar surface, could be conveniently isolated for further investigation. In this study, we report the successful culture of B. hominis cells as colonies on solid agar. Colonies were enumerated and the efficiency of plating was determined. It was observed that B. hominis could be easily cultured on agar as clones. The colonies were dome-shaped and mucoid and could grow to 3 mm in diameter. Flow cytometric analyses revealed that parasite colonies remained viable for up to 2 weeks. Viable colonies were conveniently expanded in liquid or solid media. Scanning electron microscopy revealed that each colony consists of two regions; a dome-shaped, central core region and a flattened, peripheral region. Older colonies possessed numerous strand-like surface coat projections. This study provides the first report of clonal growth of B. hominis on agar and a simple, effective method for cloning and expansion of B. hominis cells.
    Experimental Parasitology 10/2000; 96(1):9-15. · 2.15 Impact Factor
  • K Suresh, J Howe, G C Ng, L C Ho, N P Ramachandran, A K Loh, E H Yap, M Singh
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    ABSTRACT: A non-axenic and an axenic isolate of Blastocystis hominis have been induced to form cysts in vitro using an encystation medium. The morphology of the parasite at different time points was observed by scanning electron microscopy. In day-2 cultures the cysts were spherical and had a non-uniform, coarse outer surface around the body. A deep, pore-like opening was seen in some of the parasites. Most of the cysts from day-4 and day-6 cultures ruptured, revealing small, uniformly sized spherical bodies occurring in grape-like clusters. Acridine orange staining confirmed that these bodies were the progeny of Blastocystis hominis. A multiple fission-like reproduction process giving rise to many daughter Blastocystis occurs within the cyst.
    Parasitology Research 02/1994; 80(6):523-7. · 2.85 Impact Factor
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    ABSTRACT: The morphological changes occurring in Blastocystis hominis at different time points following in vitro encystment were studied by electron microscopy. The following stages of the parasite were sequentially seen: (a) the amoebic form, which was irregular in shape, with a majority of the organelles being concentrated at the condensed cytoplasmic region; (b) the pre-cystic form, which was rounded and had an electron-dense material forming a homogeneous wall around the central body; and (c) the cystic form, which had a very prominent, thick osmiophilic electron-dense wall, within which there were many inclusions and possibly reproductive granules. The amoebic form appeared to be an intermediate stage between the vacuolar form and the pre-cystic form, as this stage allowed the parasite to ingest bacteria to enhance encystment. The pre-cystic stage had previously been shown in experimental infection to be infective. The role of the cystic stage in producing infection is currently being investigated.
    Parasitology Research 02/1994; 80(4):327-35. · 2.85 Impact Factor
  • K Suresh, G C Ng, N P Ramachandran, L C Ho, E H Yap, M Singh
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    ABSTRACT: Cultures of Blastocystis hominis were induced to encyst using three encystation media: (a) an encystation medium (EM) comprising yeast extract in buffered saline containing 50% horse serum, (b) an encystation medium (CEM) comprising EM conditioned with bacterial soluble products and (c) an encystation medium (TEM) containing 0.5% trypticase in EM. Two isolates of B. hominis were studied, an axenized isolate C and a non-axenized isolate MS. In EM, isolate C did not encyst, whereas 6.1% of isolate MS had encysted by day 1. However, in CEM and TEM, 17.4% and 25.7% of isolate C, respectively, had encysted by day 5. In all three media, isolate MS encysted more readily than isolate C, with as much as 91.7% of the former encysting in TEM. As viewed by phase-contrast microscopy, cyst-like stages appeared highly refractile. Direct stool examination of juvenile Wistar rats infected with 10,000 cyst-like stages of both C and MS isolates showed Blastocystis at day 2 post-infection. At autopsy on day 7, large numbers of Blastocystis were seen in the cecum, with smaller numbers being observed in the large intestine. In contrast, rats fed with various inocula of the vacuolar stages of isolates C and MS did not become infected, indicating the importance of the encysted stages in the transmission of the parasite.
    Parasitology Research 02/1993; 79(6):456-60. · 2.85 Impact Factor

Publication Stats

74 Citations
10.71 Total Impact Points

Institutions

  • 1993–2000
    • National University of Singapore
      • Department of Microbiology
      Singapore, Singapore