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Updated: May 2, 2026
A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms
Published on: October 21, 2010
Standardisation of high throughput microdilution antifungal susceptibility testing for Candida albicans and
Holly E E Floyd1, Angela M Kavanagh1, Gabrielle J Lowe1
1Community for Open Antimicrobial Drug Discovery, Centre for Superbug Solutions, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia.
Abstract:
The Clinical and Laboratory Standards Institute (CLSI) M27 guidelines are the recommended and most commonly used protocols for broth microdilution antifungal susceptibility testing of yeasts. However, these guidelines are limited to the use of 96-well assay plates, limiting assay capacity. With the increased risk of fungal resistance emerging in the community, it is important to have alternative protocols available, that offer higher throughput and can screen more than eight to ten potential antifungal compounds per plate. This study presents an optimised broth microdilution minimum inhibitory concentration (MIC) method for testing the susceptibility of yeasts in an efficient high throughput screening setup, with minimal growth variability and maximum reproducibility. We extend the M27 guidelines and optimise the conditions for 384-well plates. Validation of the assay was performed with ten clinically used antifungals (fluconazole, amphotericin B, 5-fluorocytosine, posaconazole, voriconazole, ketoconazole, itraconazole, caspofungin diacetate, anidulafungin and micafungin) against Candida albicans and Cryptococcus neoformans.
Insights
This study optimized the Clinical and Laboratory Standards Institute (CLSI) M27 broth microdilution method for 384-well plates, enabling higher throughput antifungal susceptibility testing for yeasts.
Area of Science:
- Microbiology
- Mycology
- Pharmacology
Background:
- The Clinical and Laboratory Standards Institute (CLSI) M27 guidelines are standard for yeast antifungal susceptibility testing.
- Current M27 protocols utilize 96-well plates, limiting throughput for screening antifungal compounds.
- Emerging antifungal resistance necessitates higher-throughput methods for broader screening.
Purpose of the Study:
- To optimize the broth microdilution method for 384-well plates for high-throughput yeast antifungal susceptibility testing.
- To ensure minimal growth variability and maximum reproducibility in the adapted assay.
- To provide an efficient screening platform for potential antifungal agents.
Main Methods:
- Adapted the CLSI M27 broth microdilution protocol for use with 384-well assay plates.
- Optimized assay conditions for the larger plate format.
- Validated the optimized method using ten clinically relevant antifungal drugs against Candida albicans and Cryptococcus neoformans.
Main Results:
- Successfully adapted the M27 broth microdilution assay for 384-well plates.
- Achieved high throughput capacity for antifungal susceptibility testing.
- Demonstrated minimal growth variability and high reproducibility in the optimized assay.
Conclusions:
- The optimized 384-well plate broth microdilution method offers a high-throughput, reproducible, and efficient approach for yeast antifungal susceptibility testing.
- This adapted protocol expands upon CLSI M27 guidelines, facilitating broader screening of antifungal compounds.
- The method is validated for clinical antifungal agents against key yeast pathogens.
