A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Summary

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Abstract

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

Protocol

1. Preparation of C. albicans

C. albicans is a Risk Group 1/BSL1 microorganism. Always remember to use good aseptic/sterile techniques for work with this microorganism and follow institutional procedures for proper disposal of biohazard materials.

1. Prepare an overnight culture of C. albicans in YPD (Yeast Peptone Dextrose) liquid medium by inoculating a single colony of C. albicans into 25 mL of YPD.
2. Incubate culture in an orbital shaker (about 180 rpm) at 30 °C overnight. Most C. albicans strains will grow as yeast cells under these conditions.
3. Centrifuge ....

Discussion

Here we describe a simple, rapid, economical and highly reproducible 96 well microtiter plate model for the formation of Candida biofilms coupled with a colorimetric method that measures the metabolic activities of cells within the biofilm using XTT. This 96 well microtiter plate model for biofilm formation was originally developed for C. albicans but can be used for other Candida spp. and easily adapted for other fungal organisms. The method can be used to examine multiple parameters and facto.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sabouraud-dextrose agar	BD Biosciences	211584	To prepare plates for fresh subcultures of fungal isolates
YPD: Yeast peptone dextrose	US Biological	Y2076	Medium for propagation of overnight liquid cultures
RPMI-1640 without sodium bicarbonate supplemented with L-glutamine	Cellgro	50-020-PB	Liquid medium for biofilm formation
Morpholinepropanesulfonic acid (MOPS)	Fisher Scientific	BP308	To buffer RPMI 1640
Phosphate buffered saline, PBS	Sigma-Aldrich	P4417	Buffer for washes
XTT sodium salt	Sigma-Aldrich	X4251	See above for preparation instructions
Ringer’s lactate	Hospira Inc.	NDC0409-7953-09	For preparation of XTT solution
Menadione	Sigma-Aldrich	M5625	Caution: hazardous by skin contact, inhalation or ingestion
Petri dishes	Fisher Scientific	08-757-12
15 ml conical centrifuge tubes	Corning	430790
50 ml conical centrifuge tubes	Corning	430828
96 well microtiter plates: Polystyrene, flat-bottomed, tissue culture treated	Corning	3595
Multichannel pipette and tips	Eppendorf
Incubator	Any Supplier
Microtiter plate reader	Any Supplier