Parsek, T. (3, 9, 24, 30), but fungal biofilm formation is much less studied. is known to synthesize biofilms (11, 28, 29), as is usually biofilms are significantly less susceptible to caspofungin and amphotericin B than are planktonic cells (19). The cells within the biofilm are also resistant to the TAPI-1 actions of fluconazole and voriconazole and various microbial oxidants and peptides (17, 19). Bacterial and fungal biofilms form readily on prosthetic materials, which poses a tremendous risk of chronic contamination (10, 13, 15, 27). biofilms can form on a range of surfaces, including glass, polystyrene, and polyvinyl, and material devices, such as catheters (16). can form biofilms around the ventriculoatrial shunts used to decompress intracerebral pressure in patients with cryptococcal meningoencephalitis (32). The polysaccharide capsule of is essential for biofilm formation (18), and biofilm formation entails the shedding and accumulation of large amounts of GXM into the biofilm extracellular matrix (16). Previously, we reported that antibody to GXM in answer could inhibit biofilm formation through a process that presumably entails interference with polysaccharide shedding (18, 20). However, the effect of antibody-mediated immobilization of cells on cryptococcal biofilm formation has not been explored. In this paper, we statement that this monoclonal antibody (MAb) 18B7, which is specific for the capsular polysaccharide GXM, can capture and immobilize to surfaces, a process that promotes biofilm formation. Interestingly, we recognized planktonic variant cells that appeared to escape from your biofilm, but whose functions are not known. The results provide new insights on biofilm formation. MATERIALS AND METHODS Yeast strains and culture conditions. var. strain H99 was obtained from Mauricio del Poeta (Charleston, NC). Strains were produced in Sabouraud dextrose broth at 30C with agitation (150 to 180 rpm). was killed by heating in a 65C water bath for 30 min. Time-lapse microscopy. Poly-d-lysine glass bottom culture dishes (Ashland, MA) were coated with 10 g/ml MAb 18B7 PIP5K1A to capsule component GXM or MOPC-21, an irrelevant isotype-matched control MAb that does not bind cells were collected by centrifugation at 10,600 for 30 s, washed three times with PBS, and resuspended in media used to induce biofilm formation, termed inducing media (10% Sabouraud dextrose broth diluted in 50 mM MOPS [morpholinepropanesulfonic acid] [pH 7.5]), and a total of 2 105 cells were added to the culture dish. Live imaging was performed using an Axiovert 200 M inverted microscope and photographed with an AxioCam MRm video TAPI-1 camera controlled by the Axio Vision 4.6 software (Carl Zeiss Micro Imaging, New York, NY). Imaging was performed at 4-min intervals, using a 10 or 20 (numerical optovar of 1 1.6) objective. Immunofluorescence microscopy. biofilms were incubated for 10 h at room heat with Alexa Fluor 488-labeled MAb 18B7 (10 g/ml) and then washed with PBS. Fluorescence microscopy was performed using an Axiovert 200 M inverted microscope (10 objective, numerical optovar of 1 1.6) using green fluorescent light. Measurement of biofilm growth by XTT reduction assay. To induce biofilm formation, sterile 96-well polystyrene enzyme-linked immunosorbent assay (ELISA) plates were coated with 100 l (10 g/ml) of either MAb 18B7 or MOPC-21 and incubated at room heat for 2 h. Microtiter wells made up of heat-killed were included as unfavorable controls. Assays were carried out in six wells, TAPI-1 thus yielding six repetitions. Wells were washed three times with 0.05% Tween 20 in PBS (PBS-T). cells were harvested as explained above and resuspended in inducing media, and 1 106 cells were added to the wells. Plates were incubated at 37C for 2, 4, 6, 8, or 12 h to induce biofilm formation. Following incubation, wells were washed in triplicate with PBS-T, to remove any planktonic cells. A semiquantitative measurement of biofilm formation was obtained from the 2 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. For each well, 50 l of XTT salt answer (1 mg/ml in PBS) and 4 l of menadione answer (1 mM in acetone) were added. The colorimetric switch was measured using a microtiter reader at 492 nm. In prior studies we have exhibited that the XTT reduction assay measurements correlate with biofilm fungal cell number (16). Spot ELISA. Sterile 96-well polystyrene ELISA plates were coated with 50 l of MAb 18B7 or MOPC-21 (10 g/ml) and incubated for 1 h at.