1887

Abstract

Several techniques have been used to quantify the cytotoxicity produced by bacilli on cell monolayers; however, they are semi-quantitative or time consuming. Herein, a method based on crystal violet (CV) uptake by THP-1 cell monolayers is described. This colorimetric method quantifies the cytotoxic effect as a function of the number of remaining cells after the infection with . Since this micro-organism is not stained by the dye, it does not produce a background that affects absorbance readings. As determined by CV assay (CVA), strain H37Rv destroyed 10.5 % of THP-1 cell monolayers at 24 h and 50.52 % at 72 h, while strains lacking the complete phospholipase C locus produced a reduced cytotoxic effect. The damage estimated by microscopy corresponded to the effect quantified by CVA. The results show that the use of CVA is a rapid, sensitive and reliable quantitative assay to measure the cytotoxicity of different strains.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/jmm.0.46915-0
2007-06-01
2019-10-14
Loading full text...

Full text loading...

/deliver/fulltext/jmm/56/6/733.html?itemId=/content/journal/jmm/10.1099/jmm.0.46915-0&mimeType=html&fmt=ahah

References

  1. Carter, G., Wu, M., Drummond, D. C. & Bermudez, L. E. ( 2003; ). Characterization of biofilm formation by clinical isolates of Mycobacterium avium. J Med Microbiol 52, 747–752.[CrossRef]
    [Google Scholar]
  2. Castro-Garza, J., Quinn, F. D. & King, C. H. ( 1997; ). Assay to assess the cytotoxic effect of Mycobacterium tuberculosis and Mycobacterium bovis BCG on human alveolar pneumocytes. Abstract no. A-50 in Abstracts of the ASM Conference on Tuberculosis: Past, Present and Future, 1997. Copper Mountain, CO: American Society for Microbiology.
  3. Castro-Garza, J., King, C. H., Swords, W. E. & Quinn, F. D. ( 2002; ). Demonstration of spread by Mycobacterium tuberculosis bacilli in A549 epithelial cell monolayers. FEMS Microbiol Lett 212, 145–149.[CrossRef]
    [Google Scholar]
  4. Danelishvili, L., McGarvey, J., Li, Y. J. & Bermudez, L. E. ( 2003; ). Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells. Cell Microbiol 5, 649–660.[CrossRef]
    [Google Scholar]
  5. Daniel, A. K., Lee, R. E., Portaels, F. & Small, P. L. ( 2004; ). Analysis of Mycobacterium species for the presence of a macrolide toxin, mycolactone. Infect Immun 72, 123–132.[CrossRef]
    [Google Scholar]
  6. Dobos, K. M., Spotts, E. A., Quinn, F. D. & King, C. H. ( 2000; ). Necrosis of lung epithelial cells during infection with Mycobacterium tuberculosis is preceded by cell permeation. Infect Immun 68, 6300–6310.[CrossRef]
    [Google Scholar]
  7. Ferrari, M., Chiara, F. M. & Isetta, A. M. ( 1990; ). MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods 131, 165–172.[CrossRef]
    [Google Scholar]
  8. Fischer, L. J., Quinn, F. D., White, E. & King, C. H. ( 1996; ). Intracellular growth and cytotoxicity of Mycobacterium haemophilum in a human epithelial cell line (Hec-1-B). Infect Immun 64, 269–276.
    [Google Scholar]
  9. Franzblau, S. G., Witzig, R. S., McLaughlin, J. C., Torres, P., Madico, G., Hernandez, A., Degnan, M. T., Cook, M. B., Quenzer, V. K. & other authors ( 1998; ). Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate Alamar Blue assay. J Clin Microbiol 36, 362–366.
    [Google Scholar]
  10. Gillies, R. J., Didier, N. & Denton, M. ( 1986; ). Determination of cell number in monolayer cultures. Anal Biochem 159, 109–113.[CrossRef]
    [Google Scholar]
  11. Gomez-Flores, R., Gupta, S., Tamez-Guerra, R. & Mehta, R. T. ( 1995; ). Determination of MICs for Mycobacterium avium-M. intracellulare complex in liquid medium by a colorimetric method. J Clin Microbiol 33, 1842–1846.
    [Google Scholar]
  12. Harhaji, L., Vuckovic, O., Miljkovic, D., Stosic-Grujicic, S. & Trajkovic, V. ( 2004; ). Iron down-regulates macrophage anti-tumour activity by blocking nitric oxide production. Clin Exp Immunol 137, 109–116.[CrossRef]
    [Google Scholar]
  13. Ishiyama, M., Shiga, M., Sasamoto, K., Mizoguchi, M. & He, P. ( 1993; ). A new sulfonated tetrazolium salt that produces a highly water-soluble formazan dye. Chem Pharm Bull (Tokyo) 41, 1118–1122.[CrossRef]
    [Google Scholar]
  14. McDonough, K. A. & Kress, Y. ( 1995; ). Cytotoxicity for lung epithelial cells is a virulence-associated phenotype of Mycobacterium tuberculosis. Infect Immun 63, 4802–4811.
    [Google Scholar]
  15. Petrini, B. ( 2006; ). Mycobacterium abscessus: an emerging rapid-growing potential pathogen. APMIS 114, 319–328.[CrossRef]
    [Google Scholar]
  16. Ranger, B. S., Mahrous, E. A., Mosi, L., Adusumilli, S., Lee, R. E., Colorni, A., Rhodes, M. & Small, P. L. ( 2006; ). Globally distributed mycobacterial fish pathogens produce a novel plasmid-encoded toxic macrolide, mycolactone F. Infect Immun 74, 6037–6045.[CrossRef]
    [Google Scholar]
  17. Raynaud, C., Guilhot, C., Rauzier, J., Bordat, Y., Pelicic, V., Manganelli, R., Smith, I., Gicquel, B. & Jackson, M. ( 2002; ). Phospholipases C are involved in the virulence of Mycobacterium tuberculosis. Mol Microbiol 45, 203–217.[CrossRef]
    [Google Scholar]
  18. Read, J. K., Heggie, C. M., Meyers, W. M. & Connor, D. H. ( 1974; ). Cytotoxic activity of Mycobacterium ulcerans. Infect Immun 9, 1114–1122.
    [Google Scholar]
  19. Rothman, S. W. ( 1986; ). Technique for measuring 50 % end points in cytotoxicity assays for Clostridium difficile toxins. J Clin Pathol 39, 672–676.[CrossRef]
    [Google Scholar]
  20. Shaik, M. S., Chatterjee, A. & Singh, M. ( 2004; ). Effects of monensin liposomes on the cytotoxicity, apoptosis and expression of multidrug resistance genes in doxorubicin-resistant human breast tumour (MCF-7/dox) cell-line. J Pharm Pharmacol 56, 899–907.[CrossRef]
    [Google Scholar]
  21. Takii, T., Hamasaki, S., Hirano, K., Abe, C. & Onozaki, K. ( 2005; ). Simple fibroblast-based assay to test pyrazinamide susceptibility of Mycobacterium tuberculosis. Antimicrob Agents Chemother 49, 804–807.[CrossRef]
    [Google Scholar]
  22. Thomas, M., Finnegan, C. E., Rogers, K. M., Purcell, J. W., Trimble, A., Johnston, P. G. & Boland, M. P. ( 2004; ). STAT1: a modulator of chemotherapy-induced apoptosis. Cancer Res 64, 8357–8364.[CrossRef]
    [Google Scholar]
  23. Torrado, E., Fraga, A. G., Castro, A. G., Stragier, P., Meyers, W. M., Portaels, F., Silva, M. T. & Pedrosa, J. ( 2007; ). Evidence for an intramacrophage growth phase of Mycobacterium ulcerans. Infect Immun 75, 977–987.[CrossRef]
    [Google Scholar]
  24. Trifiro, S., Bourgault, A. M., Lebel, F. & Rene, P. ( 1990; ). Ghost mycobacteria on Gram stain. J Clin Microbiol 28, 146–147.
    [Google Scholar]
  25. Trucksis, M., Pritchett, C. L. & Reimschuessel, R. ( 2005; ). Mycobacterium marinum and fish and frog models of infection. In Tuberculosis and the Tubercle Bacillus, pp. 529–535. Edited by S. T. Cole, K. D. Eisenach, D. N. McMurray & W. R. Jacobs, Jr. Washington, DC: American Society for Microbiology.
  26. Vera-Cabrera, L., Molina-Torres, C., Hernández-Vera, M. A., Barrios-García, H. B., Balckwood, K., Villarreal-Treviño, L., Ocampo-Candiani, J., Welsh, O. & Castro-Garza, J. ( 2007; ). Genetic characterization of Mycobacterium tuberculosis clinical isolates with deletions in the plcA-plcB-plcC locus. Tuberculosis (Edinb) 87, 21–29.[CrossRef]
    [Google Scholar]
  27. Zivadinovic, D., Gametchu, B. & Watson, C. S. ( 2005; ). Membrane estrogen receptor-alpha levels in MCF-7 breast cancer cells predict cAMP and proliferation responses. Breast Cancer Res 7R101R–112.[CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/jmm.0.46915-0
Loading
/content/journal/jmm/10.1099/jmm.0.46915-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error