OD600 Calculators
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OD600 and Cell Density
E. coli in LB medium: OD600 = 1.0 ≈ 8 × 10⁸ cells/mL (CAUTION: this varies by strain, instrument, cuvette path length, and growth phase). Always calibrate OD600 vs. actual CFU/mL for your specific conditions. OD600 measures all particles — live, dead, debris — not just viable cells.
Linear Range
Most spectrophotometers: Beer-Lambert linear range OD600 ≤ 0.3–0.4. Above this: multiple light scattering → readings underestimate true cell density. For OD > 0.4: dilute sample (e.g., 10-fold) and multiply reading by dilution factor. Never use OD readings above 0.8 without dilution for quantitative work.
Growth Rate from OD600
Plot ln(OD600) vs. time on a graph. During exponential phase: linear relationship. μ = slope = [ln(OD₂) − ln(OD₁)] / (t₂ − t₁). Doubling time = 0.693 / μ. Example: OD₆₀₀ from 0.10 to 0.80 in 3 hours: μ = (ln 0.80 − ln 0.10) / 3 = 2.08/3 = 0.693 h⁻¹; t_d = 0.693/0.693 = 1 hour.
Practical Tips
Always blank with uninoculated medium. Use cuvettes with 1 cm path length. Measure at the same time each reading for growth curves. OD600 = 0.6–0.8 = mid-log phase for E. coli (good for induction or experiments).
Glossary
Frequently Asked Questions
OD600 = absorbance of a microbial culture at 600 nm. Based on Beer-Lambert law: A = ε × c × l; absorbance is proportional to the concentration of scattering particles (bacterial cells). At 600 nm: sufficient absorption for sensitivity; minimal interference from colored media components. OD600 measures all particles — live, dead, debris — and cannot distinguish dead from live cells. For E. coli in LB at 37°C: OD600 ≈ 1.0 corresponds to ~8 × 10⁸ cells/mL (rule of thumb; always verify for your strain). OD600 is the most common rapid bacterial density measurement — fast (< 1 min), non-destructive, and requires minimal sample volume.
Plot ln(OD600) vs. time. During exponential phase: the plot is linear. Specific growth rate: μ = [ln(OD₂) − ln(OD₁)] / (t₂ − t₁). Units: h⁻¹ (or min⁻¹). Doubling time: t_d = ln(2)/μ = 0.693/μ. Example: time points 0 h and 2 h; OD600 = 0.05 and 0.40: μ = (ln(0.40) − ln(0.05)) / 2 = (−0.916 − (−2.996)) / 2 = 2.08/2 = 1.04 h⁻¹. t_d = 0.693/1.04 = 0.67 h = 40 min. IMPORTANT: only use data points from the exponential phase (linear portion of semi-log plot) — lag and stationary phases give incorrect μ values.
Beer-Lambert law: A = ε × c × l is linear when absorbance is proportional to concentration. For OD600: linear range is typically OD ≤ 0.3–0.4 in most spectrophotometers. Above this: multiple scattering events → some scattered photons reach the detector → apparent lower OD → underestimation of true cell density. Consequence: if OD600 = 0.8 is measured without dilution, the true value may be 1.0 or higher. Rule: for accurate measurements, dilute cultures above OD 0.4 and multiply the measured OD by the dilution factor. Modern instruments with narrow-beam optics may be linear to OD 0.8–1.0, but always verify for your specific instrument.
OD600 errors: Not blanking: always zero the spectrophotometer with blank medium before measuring cultures. Bubbles in cuvette: trapped air bubbles scatter light → falsely elevated OD; ensure cuvette is bubble-free. Condensation on cuvette: wipe cuvette sides clean before measuring. Debris or aggregates: large particles scatter more; clumped cultures give unreliable OD. Path length: ensure cuvette path length is 1 cm (standard); non-standard path lengths require correction. Measuring above linear range: dilute cultures if OD > 0.4. Temperature effects: OD600 is not strongly temperature-dependent for typical ranges, but keep samples at similar temperatures for consistent results. Cell morphology changes: stressed cells, elongated cells, or other morphological changes can alter the OD-to-cell-count relationship.