Confocal Microscopy of Biofilm Formation

The strains being analyzed were grown overnight in YPD at 30°C. The overnight cultures were used to inoculate wells with 2 mls of fresh YPD media at an OD₆₀₀ of 0.5 on silicone squares (Bentec Medical Inc.) that were pretreated with fetal bovine serum (FBS). The cells were allowed to adhere to the silicone for 90 min in an incubator-shaker at 37°C and 60 rpm. Following the adherence, the squares were washed in PBS to remove any nonadherent cells and placed in wells containing 2 mls of new YPD media. Biofilm imaging is adapted from [79 (link)] with modifications. After 48 hours, the biofilms were fixed using 4% formaldehyde and 1.5% glutaraldehyde in 1xPBS on an orbital mixer for 1 hour. After fixation the specimens were washed in 1xPBS. The fixed biofilms were stained with concanavalin A, Alexa Fluor 594 Conjugate (Life Technologies) at a concentration of 25 μg/ml in PBS for two days on an orbital mixer. The fixed and stained biofilms on the silicone squares were then transferred to glass scintillation vials. To dehydrate the samples, 2 mls of methanol were added to the samples and allowed to infiltrate on an orbital mixer for 20 min. The methanol was aspirated out and 2 mls of methanol were briefly added. After the 100% methanol addition, a 50:50 mixture of methanol and methyl salicylate was added. The 50:50 mixture was aspirated out and replaced with 100% methyl salicylate. The vials were gently agitated until the samples were completely cleared through the matching of refractive index. In order to image these samples using an inverted confocal microscope and avoiding the use of plastic, a cover glass was cemented to the bottom of a black-anodized aluminum stage insert. A silicone ring (thickness 300μm) and a small amount of methyl salicylate were added to this constructed well and the biofilm was placed on the ring with the apical side facing down, using the surface tension between the methyl salicylate and the silicone square to hold the silicon in place. The biofilms were imaged using a slit-scan confocal optical unit on a Zeiss Axiovert 200 microscope. A 40x 0.85-numerical aperture oil immersion objective was used in order to provide enough working distance to focus through the full thickness of the biofilms. Optical sections were collected in several series of 130 planes with a total sum of 500 planes (Fig 4) or 557 planes (Fig 6A) at 0.9 μm step-size. The stacks were concatenated and processed using FIJI software [80 (link)]. The images were processed using the Background Subtract plugin and the final images were obtained using a resliced, maximum intensity Z-projection. The apical view projections were obtained using the Temporal Color-code plugin and the Ice lookup table.