A Modular Workflow for Quantitative, Structural and Functional Analysis of Leptospira Biofilms

We study biofilms as protective structures enabling environmental and host persistence, investigating the formation dynamics, molecular composition, adaptative features and contribution to infection. Combining crystal bio assay, time text imaging, confocal microscopy, scanning electron microscopy and transcriptomics in our labs, advances biofilm research through integrated multi dimension analysis. To begin, grow Leptospira cells in EMJH medium in flat bottomed screw cap glass tubes.

Incubate the cultures under aerobic conditions at 30 degrees Celsius without shaking until they reach mid logarithmic phase. Ensure the cultures reach an optical density between 0.2 and 0.4 at 405 nanometers, which corresponds to two to five times 10 to the power of eight cells per milliliter. Using a dark field microscope at 20 x magnification, verify that the cells are modal and not clumped.

Dilute the verified mid log arrhythmic phase culture at a one to 100 ratio in fresh EMJH medium to obtain approximately one times 10 to the power of six cells per milliliter and mix gently by inversion without vortexing. Now use sterile forceps to place one 0.1 micrometer sterile hydrophilic polycarbonate membrane flat at the bottom of each well in a sterile 24 well plate with lid. Add one milliliter of sterile EMJH medium to each well and pre-soak the membrane for two hours at 30 degrees Celsius.

Next, remove the soaking solution from each well without displacing the membrane and add 1.5 milliliters of diluted bacterial suspension, ensuring the membrane remains firmly in place at the bottom. Then, place a water-filled tray inside the incubator to maintain humidity. Incubate the plate at 30 degrees Celsius under static conditions to allow biofilm formation.

Leaving the plate for up to three weeks for slow growing strains. At the desired time point, carefully aspirate as much culture medium as possible without disturbing the biofilm. Rinse each well gently with one milliliter of sterile PBS while keeping the membrane flat against the bottom.

Add one milliliter of 4%paraform aldehyde in PBS to each well and incubate at 37 degrees Celsius for 30 minutes to fix the biofilm samples. After incubation, remove the fixative and gently rinse twice with one milliliter PBS. Add one milliliter of 0.1%weight by volume crystal violet solution to each well and incubate at room temperature for 15 minutes, making sure the cover slip is fully submerged.

Then discard the crystal violet dye from each well and rinse twice with one milliliter of PBS. Tilt the plate and drain all remaining liquid. Leave the plate at room temperature to air dry until the substrate appears completely dry, preferably overnight.

Next, add 500 microliters of elucian buffer composed of 50%ethanol and 50%glacial acetic acid by volume to each well. Pipette up and down to fully dissolve the stain bound to the biofilm. Now, transfer 200 microliters of each sample to an optically clear 96 well microplate.

Measure absorbance at 570 nanometers and subtract background values from uninoculated controls processed through all steps. Record the mean and standard deviation for at least three technical replicates. Obtain the biofilms in well plates as demonstrated earlier.

Add a solution of 4%paraform aldehyde, and 1%glutaraldehyde in 0.2 molar sodium buffer at pH 7.4 into the wells. Incubate the fixed samples for 30 minutes at 37 degrees Celsius. Remove the fixative and rinse the sample twice with PBS to preserve the surface attached biofilm while minimizing detachment.

Now immerse the cover slip in 1%osmium tetroxide diluted in PBS and incubate for one hour to enhance scanning electron microscopy contrast. Then rinse the substrate twice with PBS. Dehydrate the samples by immersing them sequentially for 10 minutes each in graded ethanol series.

Next, add 500 microliters of hexamethyldisilazane and incubate for five minutes. Then replace with fresh hexamethyldisilazane and incubate for an additional five minutes. Afterward, remove the excess solution and let the sample air dry completely under a fume hood.

Now mount the dried samples onto scanning electron microscopy stubs using double-sided conductive carbon tape. Sputter coat the samples with a thin layer, approximately 10 nanometers of gold or platinum to enhance electron contrast for imaging. Finally, load the prepared stubs into the scanning electron microscope using the appropriate sample holder.

Evacuate the chamber overnight if possible to improve imaging quality. Then acquire secondary electron images at five to 15 kilovolts using suitable magnifications to visualize the ultra structure of the biofilm. After 21 days of incubation, crystal violet staining revealed visible biofilm patterns on both polycarbonate filters and glass cover slips for leptospira interrogans and leptospira biflexa.

With each species displaying distinct architectural footprints such as dotlike, branching or reticulated forms. Absorbance measurements at 570 nanometers confirmed greater crystal violet retention in leptospira byflexa biofilms compared to leptospira interrogans across all time points indicating higher biomass accumulation in the strain. Scanning electron microscopy of leptospira interrogans biofilms captured early extracellular matrix deposits in three day old biofilms, a mature and channeled basal face with three dimensional structure at 14 days and fully developed matrix consolidation with dense architecture by 21 days.

Our optimized protocol synchronizes complementary readouts from identical cultures, increasing robustness, reducing variability and revealing dynamic and structural information and available with single method approaches. By integrating complementary analysis, our findings standardized biofilm assessment, clarify leptospira dynamics and architecture. They link structure to virulence and strengthen reproducible comparative research.

Future mutants to link regulation with biofilm morphology and dynamics, clarifying environmental persistence and evaluate strategies disrupting biofilm information or promoting dispersal.