Automated OD600 Growth-Curve Kinetics of Staphylococcus aureus Under Sub-Inhibitory Oxacillin Stress

A two-fold oxacillin series (0.03-16 µg/mL) plus a no-drug control is tested in 4 technical replicate wells per concentration across 3 biological replicates on separate days. Each 96-well plate includes a medium-only blank column (for OD blanking) and a no-drug growth control. The plate reader is set to 37 C with continuous orbital shaking and 15-min OD600 reads for 24 h. Wells are randomized across the plate to avoid positional bias; lid condensation is mitigated with an anti-fog treatment.

BSL-2 for S. aureus including MRSA; manipulate cultures in a Class II biosafety cabinet and minimize aerosols (no vortexing open tubes). PPE: lab coat, nitrile gloves, eye protection. Oxacillin is a beta-lactam — handle as a sensitizer; weigh powder with a mask in a hood and avoid inhalation/skin contact (allergy risk). Decontaminate plates and disposables by autoclaving or 10% bleach. Clean the reader chamber per institutional biosafety SOP after MRSA runs.

No-drug growth control: defines maximal µmax, lag, and yield for normalization. Medium-only blank wells: subtract abiotic OD drift and condensation. Sterility check: blank wells must remain flat (no growth). Reference QC: ATCC 29213 oxacillin endpoint must align with its CLSI QC range, validating drug potency. Vehicle control (if oxacillin dissolved in any solvent) at matched solvent concentration. A high-concentration well above MIC serves as a growth-inhibition positive control (flat curve).

The no-drug control shows a sigmoidal curve: lag ~1-3 h, exponential µmax ~0.8-1.2 h^-1 (doubling ~35-50 min in CAMHB), plateau OD600 ~0.8-1.2. Sub-inhibitory oxacillin extends lag and lowers µmax dose-dependently in MSSA; MRSA curves remain near-control until high concentrations. Blank wells stay flat (<0.05 OD drift). Technical-replicate µmax CV <15%.

• Staphylococcus aureus ATCC 29213 (MSSA) and a mecA+ MRSA comparator (e.g., ATCC 43300)
• Cation-adjusted Mueller-Hinton Broth (CAMHB) supplemented with 2% NaCl for oxacillin testing
• Oxacillin sodium salt reference standard, stock 5120 µg/mL in sterile water
• 0.5 McFarland standard / densitometer
• Sterile flat-bottom, clear 96-well plates with optically clear lids
• Breathable sterile sealing membrane or anti-condensation lid treatment
• Sterile saline (0.85% NaCl), tips, multichannel pipette, reservoirs
• Mueller-Hinton agar for inoculum CFU verification

To generate high-resolution growth curves of S. aureus by reading OD600 every 15 minutes for 18-24 h in a temperature-controlled microplate reader, and to extract quantitative kinetic parameters (lag phase duration, µmax, doubling time, carrying capacity) as a function of oxacillin concentration. This enables precise comparison of growth dynamics beyond a single endpoint MIC reading.

1. Streak S. aureus on MHA; incubate 18-24 h at 35 C.
2. Prepare a two-fold oxacillin series in 2% NaCl-CAMHB at 2x final concentrations.
3. Add 100 µL of each 2x drug dilution to assigned wells; add 100 µL drug-free medium to growth-control wells.
4. Suspend colonies to 0.5 McFarland in saline; dilute to ~1 x 10^6 CFU/mL in CAMHB.
5. Add 100 µL inoculum to each well for a final ~5 x 10^5 CFU/mL; add 200 µL medium to blank wells.
6. Plate 10 µL inoculum dilution on MHA to verify CFU.
7. Apply a clear lid/anti-fog film; load into the reader pre-warmed to 37 C.
8. Program OD600 reads every 15 min for 24 h with 30 s orbital shaking before each read.
9. Export the kinetic data; subtract the blank trajectory.
10. Save raw and blanked data for curve fitting.

Independent variable: oxacillin concentration (0.03-16 µg/mL) and strain (MSSA vs MRSA). Dependent variables: lag time, µmax (h^-1), doubling time, carrying capacity (max OD600), and area under the curve. Controlled variables: inoculum density (5 x 10^5 CFU/mL), medium (2% NaCl-CAMHB), 37 C, shaking pattern, read interval (15 min), plate type, and total run time.

Increasing sub-inhibitory oxacillin concentrations will progressively extend the lag phase and reduce the maximum specific growth rate (µmax) of methicillin-susceptible S. aureus ATCC 29213, with little effect on methicillin-resistant S. aureus (MRSA) carrying mecA until much higher concentrations, producing a quantifiable rightward shift in the dose-response of kinetic parameters.

Blank-subtract each well, then fit growth curves with a logistic or Gompertz model (R 'growthcurver' package, or Python 'gcfit'/scipy) to extract lag, µmax, doubling time, and carrying capacity. Alternatively compute µmax from the maximum slope of ln(OD) vs time over a sliding window. Average parameters across technical replicates, then biological replicates. Normalize kinetic parameters to the no-drug control and plot versus oxacillin concentration to derive an IC50 for growth rate.

1. Noisy/condensation-driven OD spikes -> apply anti-fog lid treatment, pre-warm the reader, reduce read frequency slightly, or add shaking before reads. 2. No lag separation between doses -> drug may be subpotent or NaCl supplement omitted; remake oxacillin and confirm 2% NaCl medium. 3. Poor curve fit -> ensure enough exponential-phase points; reduce read interval to 10 min. 4. Early plateau at low OD -> inoculum too low or medium-limited; verify CFU and consider richer medium. 5. Settling/clumping of S. aureus -> increase orbital shaking and ensure homogeneous inoculum before loading.

Compare µmax and lag across concentrations by one-way ANOVA with Dunnett's test against the no-drug control (alpha = 0.05); compare MSSA vs MRSA dose-response curves by two-way ANOVA (factors: strain x concentration) with Sidak correction. Fit IC50 with nonlinear regression and compare via extra-sum-of-squares F-test. n = 3 biological replicates. Power analysis targets 80% power to detect a 20% reduction in µmax at alpha 0.05.