An In Vitro Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilm

Abstract

Source: Chandley, P., et al. A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms. J. Vis. Exp. (2022).

This video demonstrates an in vitro technique to study the effect of antibodies on fungal biofilms. The pathogenic fungus Candida tropicalis secretes a virulence factor secreted aspartyl proteinase 2 (Sap2), facilitating biofilm formation. Upon introducing a heat-inactivated serum containing Sap2-specific antibodies that inhibit biofilm maturation, the viability of the fungal cells is assessed using a chromogenic assay.

Protocol

1. C. tropicalis biofilm formation Prepare Candida biofilms in a 96-well flat-bottomed polystyrene microtiter plate as described earlier (Figure 1). Add 100 µL of C. tropicalis culture (from 106 cells/mL stock, prepared as above) to a 96-well microtiter plate using a multichannel pipette (Figure 2A). Keep the last two columns (11 and 12) as 'no fungus plus …

Representative Results

Figure 1: Imaging Candida tropicalis biofilm. (A) Visualization of Candida tropicalis biofilm formed on the bottom of a 96-well microtiter plate after removal of RPMI medium, using an inverted microscope. The image was captured using brightfield microscopy (no stain was used). (B) Visualization of C. tropicalis<…

Materials

15 mL conical centrifuge tubes	BD Falcon	546021
1x PBS	–	Prepared in lab	NaCl : 4 g KCl : 0.1 g Na2HPO4: 0.72 g KH2PO4 : 0.12 g Water 500 mL. Adjust pH to 7.4
50 mL conical centrifuge tubes	BD Falcon	546041
96-well microtiter plates	Nunc	442404
Incubator	Generic
Menadione	Sigma	M5625
Microtiter Plate Reader	Generic
Multichannel pipette and tips	Generic
Petri dishes	Tarson	460090
Ringers Lactate	–	Prepared in lab	sodium chloride 0.6 g sodium lactate 0.312 g potassium chloride 0.035 g calcium chloride 0.027 g Water 100 mL. Adjust to pH 7.0
RPMI 1640 MOPS	Himedia	AT180
XTT	Invitrogen	X6493