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Evaluation of three quantitative microtiter cytotoxicity assays for Escherichia coli shiga toxin

M R Franzolin C A Menezes; F A Lima; R M F Piazza; L R Trabulsi; Congresso do Instituto de Ciências Biomédicas (3. 2001 São Paulo)

Resumos São Paulo: Comissão de Cultura e Extensão Universitária do ICB/USP, 2001

São Paulo Comissão de Cultura e Extensão Universitária do ICB/USP 2001

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  • Título:
    Evaluation of three quantitative microtiter cytotoxicity assays for Escherichia coli shiga toxin
  • Autor: M R Franzolin
  • C A Menezes; F A Lima; R M F Piazza; L R Trabulsi; Congresso do Instituto de Ciências Biomédicas (3. 2001 São Paulo)
  • Assuntos: MICROBIOLOGIA
  • É parte de: Resumos São Paulo: Comissão de Cultura e Extensão Universitária do ICB/USP, 2001
  • Descrição: Enterohemorrhagic E. coli (EHEC) has been the cause of many large diarrhea outbreaks all over the world. The disease caused by EHEC is called hemorragic colitis that is frequently associated with hemolytic uremic syndrome (HUS). Toxins produced by EHEC (Stx1 and Stx2) cause both bloody and hemolytic uremic syndrome, formerly designated Shiga-like toxins or verotoxins. Usually, the cytopathic effects of these toxins are detected by cytotoxicity assay in Vero cells, as a qualitative method. In order to compare qualitative and quantitative methods, three different stains were tested, neutral red, crystal violet and amido black. The cytotoxicity activity of E. coli was assayed by exposing several dilutions of culture supernatant to Vero cell monolayers in 96-microtiter culture plates. Stx1- or Stx2-positive E. coli O157:H7 were growth in two different culture media, centrifuged and the supernatant were filtered. Also, a French Press extract of Stx1-positive EHEC was tested. The cell monolayers were incubated for 72 hours with the samples and toxin potency was estimated by staining residual cells, followed by a visual and a spectrophotometer inspection The analyzed samples presented elevated cytotoxic effect in Vero cells as expected. On the other hand, despite the fact that LLCMK2 cells being a monkey kidney epithelial cells, when tested for cytotoxic activity, they presented low sensitivity and showed to be not adequate for this assay. The optical density obtained in
    ELISA reader of each sample was in total accordance with the visual evaluation. And,. neutral red stain showed to be more sensitive than violet crystal and amido black. Besides, the quantitative assay is suitable when several samples will be tested
  • Editor: São Paulo Comissão de Cultura e Extensão Universitária do ICB/USP
  • Data de criação/publicação: 2001
  • Formato: 1 v. pôster 189.
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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