Detection of Viable Metabolically Active Yeast Cells Using a Colorimetric Assay


Authors: F. Růžička ;  V. Holá
Authors‘ workplace: Mikrobiologický ústav LF a FN u sv. Anny v Brně
Published in: Epidemiol. Mikrobiol. Imunol. 57, 2008, č. 1, s. 4-7

Overview

The increasing concern of yeasts able to form biofilm brings about the need for susceptibility testing of both planktonic and biofilm cells. Detection of viability or metabolic activity of yeast cells after exposure to antimicrobials plays a key role in the assessment of susceptibility testing results. Colorimetric assays based on the color change of the medium in the presence of metabolically active cells proved suitable for this purpose. In this study, the usability of a colorimetric assay with the resazurin redox indicator for monitoring the effect of yeast inoculum density on the reduction rate was tested. As correlation between the color change rate and inoculum density was observed, approximate quantification of viable cells was possible. The assay would be of relevance to antifungal susceptibility testing in both planktonic and biofilm yeasts.

Key words:
yeasts – Candida – biofilm – Alamar blue – resazurin – reduction – colorimetric assay.


Sources

1. Baker, C. N., Banerjee, S. N., Tenover, F. C. Evaluation of Alamar colorimetric MIC method for antimicrobial susceptibility testing of gram-negative bacteria. J Clin Microbiol, 1994, 32, 5, 1261–1267.

2. Baker, C. N., Tenover, F. C. Evaluation of Alamar colorimetric broth microdilution susceptibility testing method for staphylococci and enterococci. J Clin Microbiol, 1996, 34, 11, 2654–2659.

3. Ceri, H., Olson, M. E., Stremick, C., Read, R. R., Morck, D., Buret, A. The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J Clin Microbiol, 1999, 37, 6, 1771–1776.

4. Douglas, L. J. Candida biofilms and their role in infection. Trends Microbiol, 2003, 11, 1, 30–36.

5. Hawser, S. P., Douglas, L. J. Resistance of Candida albicans biofilms to antifungal agents in vitro. Antimicrob Agents Chemother, 1995, 39, 9, 2128–2131.

6. Holá V., Růžička F., Tejkalová R., Votava M. Stanovení citlivosti k antibiotikům u biofilmpozitivních forem mikroorganismů. Klin Mikrobiol Infekc Lek, 2004, 10, 5, 218–222.

7. Jahn, B., Stuben, A., Bhakdi, S. Colorimetric susceptibility testing for Aspergillus fumigatus: comparison of menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and Alamar blue tests. J Clin Microbiol, 1996, 34, 8, 2039–2041.

8. Kuhn, D. M., George, T., Chandra, J., Mukherjee, P. K., Ghannoum, M. A. Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins. Antimicrob Agents Chemother, 2002, 46, 6, 1773–1780.

9. Martin, A., Camacho, M., Portaels, F., Palomino, J. C. Resazurin microtiter assay plate testing of Mycobacterium tuberculosis susceptibilities to second-line drugs: rapid, simple, and inexpensive method. Antimicrob Agents Chemother, 2003, 47, 11, 3616–3619.

10. National Committee for Clinical Laboratory Standards (NCCLS). Reference method for broth dilution testing of yeasts: Approved standard, NCCLS document M27-A2. 2. vyd. Wayne, Pensylvania: NCCLS, 2002. 29 s., ISBN 1-56238-469-4.

11. Nero, L. A., Beloti, V.; Barros, M. A. F., Santana, E. H. W., Pereira, M. S. et al. Assessment of the efficiency of SimPlate™ total plate count color indicator (TPC CI) to quantify mesophilic aerobic microorganisms in pasteurized milk. Braz J Microbiol, 2002, 33, 44–48.

12. O’Brien, J., Wilson, I., Orton, T., Pognan, F. Investigation of the Alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem, 2000, 267, 17, 5421–5426.

13. Pettit, R. K., Weber, C. A., Kean, M. J., Hoffmann, H. et al. Microplate Alamar Blue Assay for Staphylococcus epidermidis Biofilm Susceptibility Testing. Antimicrob Agents Chemother, 2005, 49, 7, 2612–2617.

14. Pfaller, M. A., Espinel-Ingroff, A., Jones, R. N. Clinical evaluation of the Sensititre YeastOne colorimetric antifungal plate for antifungal susceptibility testing of the new triazoles voriconazole, posaconazole, and ravuconazole. J Clin Microbiol, 2004, 42, 10, 4577–4580.

15. Pfaller, M.A., Vu, Q., Lancaster, M., Espinel-Ingroff, A. et al. Multisite reproducibility of colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates. J Clin Microbiol, 1994, 32, 7, 1625–1628.

16. Ramage, G., Walle, K. V., Wickes, B. L., López-Ribot, J. L. Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother, 2001, 45, 9, 2475–2479.

17. Rasmussen, E. V. Use of fluorescent redox indicators to evaluate cell proliferation and viability. In Vitro Mol Toxicol, 1999, 12, 47–58.

18. To, W. K., Fothergill, A. W., Rinaldi, M. G. Comparative evaluation of macrodilution and alamar colorimetric microdilution broth methods for antifungal susceptibility testing of yeast isolates. J Clin Microbiol, 1995, 33, 10, 2660–2664.

Labels
Hygiene and epidemiology Medical virology Clinical microbiology
Login
Forgotten password

Don‘t have an account?  Create new account

Forgotten password

Enter the email address that you registered with. We will send you instructions on how to set a new password.

Login

Don‘t have an account?  Create new account