TCID50 Calculator (Spearman–Kärber & Reed–Muench)

How it works

Spearman–Kärber: log10 endpoint = x₁ − d/2 + d × Σ(positive ÷ wells), where x₁ = log10 of the first dilution’s reciprocal and d = log10 dilution step · Reed–Muench: interpolate where cumulative % infected crosses 50% · TCID50/mL = 10^endpoint ÷ inoculum volume (mL) · PFU/mL ≈ 0.69 × TCID50/mL

The 50% tissue culture infectious dose (TCID50) is the virus dilution that infects half of the inoculated cultures. You inoculate replicate wells with a serial dilution, score each well for cytopathic effect, and estimate the 50% endpoint. The Spearman–Kärber method sums the fraction infected at each dilution and is the more accurate of the classic methods when the first dilution is fully positive and the last fully negative; the Reed–Muench method (1938) interpolates between the dilutions either side of 50% using cumulative counts. Dividing by the inoculum volume gives TCID50 per mL. By the Poisson distribution, one TCID50 corresponds to about 0.69 plaque-forming units.

Worked example

Eight wells per 10-fold dilution from 10⁻¹, inoculum 0.1 mL, with 8, 8, 8, 6, 3, 0, 0 and 0 positive wells. Σ fraction = 1 + 1 + 1 + 0.75 + 0.375 = 4.125, so the endpoint is 1 − 0.5 + 4.125 = 4.625 (10^−4.63). Per mL that is 10^(4.625 + 1) = 4.22 × 10⁵ TCID50/mL (log10 5.63); Reed–Muench gives 4.11 × 10⁵ and the estimated titre is 2.91 × 10⁵ PFU/mL.

Sources: Lei C et al. On the calculation of TCID50 for quantitation of virus infectivity. Virol Sin 2021;36:141–4 · Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Hyg 1938;27:493–7

Frequently asked questions