From hemocytometer or OD600 readings.
Hemocytometer: cells/mL = (cells counted / squares counted) × dilution factor × 10⁴. The standard hemocytometer grid volume is 0.1 μL per large square. Count cells in at least 4-5 squares for statistical reliability. For accurate counts, aim for 20-50 cells per large square — dilute or concentrate accordingly. OD600 method: for E. coli, OD600 of 1.0 ≈ 8 × 10⁸ cells/mL (this varies by species and growth phase). Trypan blue exclusion distinguishes live (clear) from dead (blue) cells. Automated cell counters (Countess, TC20) use image analysis for faster, more reproducible counting. Cell density is critical for seeding experiments, passage calculations, and transfection protocols.