Brief Communication: Confocal microscopy of oral streptococcal biofilms grown in simulated microgravity using a random positioning machine

Streptococcal strains and culture conditions

S. mutans UA159 (constitutively expressing superfolder GFP in pDL278) and S. gordonii DL1 (constitutively expressing DsRed-Express2 in pDL278) were used in all experiments, previously described in²⁵ and obtained from Dr. Robert Shields (Arkansas State University). For each biofilm experiment, strains were streaked for single colony isolation from −80 °C stocks (40% (v/v) glycerol) onto Brain Heart Infusion (BHI) agar plates containing 1 mg/ml spectinomycin. After 48 h of growth at 37 °C and 5% CO₂, a single colony of each strain was inoculated from these plates into culture tubes containing BHI broth + 1 mg/ml spectinomycin and grown statically for 16-18 h at 37 °C and 5% CO₂.

Inoculation and setup of RPM biofilms

The overall methodology and experimental design are presented in Fig. 1. All steps of biofilm inoculation and setup were performed in a class 2AII biosafety cabinet to maintain sterility. For single-species biofilms, each overnight culture was diluted to an OD₆₀₀ = 0.05 in semi-defined biofilm-promoting medium²⁶ containing 10 mM sucrose, 11 mM glucose, and 500 µg/ml spectinomycin. For dual-species biofilms, overnight cultures of S. mutans and S. gordonii were each diluted to an OD₆₀₀ = 0.025 into the same tube of biofilm medium. Diluted inoculums were vortexed for 10 sec, and ~390 µl aliquots were immediately transferred to replicate wells of a 96-well sterile glass-bottomed sensoplate (Greiner Bio-One), as this volume consistently yielded zero head space in the wells. A silicone micro-mat (cut to a size that covered all wells filled with inoculum) (Thermo Scientific) was sterilized with 70% (v/v) ethanol, dried completely, and used to seal the inoculum-filled wells. After ensuring there were no leaks or major air bubbles in the wells, each plate was wrapped in parafilm and placed in a plastic sealed bag prior to placing it on the RPM. All RPM experiments were conducted using Airbus RPM 2.0 instruments housed at the Kennedy Space Center Microgravity Simulation Support Facility (KSC-MSSF). Plates were grown on RPMs using the “partial G motion mode” set for either a 0 × g or a 0.9 × g path for 22-24 h at 37 °C and 5% CO₂. As a 1 × g (normal gravity) control, biofilm plates were grown statically in the same incubator for the same period.

Confocal microscopy and analysis of biofilm images

After growth, biofilms were immediately imaged using an inverted stage Nikon A1R point scanning confocal microscope with a Plan Fluor 40x Oil DIC H N2 lens. Green fluorescence was detected with 488 nm excitation and 535 nm emission, and red fluorescence was detected with 561 nm excitation and 595 nm emission. At least n = 3 independent experiments were conducted for both single species and dual species biofilms at 1 × g (normal gravity), simulated 0.9 × g (RPM), and simulated 0 × g (RPM), with n = 13-28 total random fields of view acquired for each bacterial species and biofilm growth condition. Biofilms were analyzed for biovolume using Nikon NIS-Elements Imaging Software, followed by statistical analysis using Sigmaplot 14.

Quantification of biofilm cell viability

For CFU determinations, parallel 0 ×g RPM and 1 ×g biofilm cultures were grown as described above (duplicate wells per strain). After approximately 20.5 h growth, the silicone sealing mat was carefully removed from each 96-well plate. The entire content of one set of wells was harvested by scraping and vigorous pipetting to collect both attached and planktonic cells (“total biofilm well”). The duplicate set of wells was used to enumerate viable cells in the attached biofilm, whereby the culture supernatant (containing planktonic bacteria) was removed, an equal volume of sterile media was added, followed by harvesting of attached cells by scraping and vigorous pipetting (“attached biofilm well”). Statistical analysis was performed using Graphpad Prism 10.