TCID50 Virus Titer Calculator

Calculate virus titer in TCID50/mL using Reed-Muench or Spearman-Karber method.

Log10 dilution where 50% CPE (interpolated) (log10 dilution)

Volume inoculated per well (mL) (mL)

Observed % CPE at dilution above 50% (%)

Observed % CPE at dilution below 50% (%)

Log10 dilution step

Usually 1 for 10-fold dilutions

Please check your inputs and try again.

TCID50 Versus PFU

Both measure infectious virus but by different endpoints. TCID50 is a statistical estimate — the dilution infecting half the wells — while PFU counts discrete plaques formed by individual infectious particles.

	TCID50	PFU
Endpoint	Any CPE in the well	Discrete plaque
Requires overlay	No	Yes
Works for non-lytic virus	Sometimes	Often not
Conversion	—	PFU ≈ 0.7 × TCID50

The 0.7 conversion comes from Poisson statistics: a well showing CPE may have received one particle or several, and the factor accounts for that. It is an approximation, and virus-specific validation is preferable to assuming it.

Reed–Muench and Spearman–Kärber

Both estimate the 50% endpoint by interpolating between the dilutions bracketing it, since no dilution gives exactly 50% in practice.

Method	Approach	Note
Reed–Muench	Cumulative totals across dilutions	Historically dominant
Spearman–Kärber	Weighted mean of transition	Gives confidence intervals

Spearman–Kärber is generally preferred now because it yields a variance estimate, so results can carry error bars. Reed–Muench gives a point estimate only.

Why the Volume Correction Matters

The endpoint dilution gives TCID50 per inoculum volume, not per mL. Dividing by the volume inoculated converts it — with 0.1 mL per well, the titre per mL is ten times the endpoint value.

Omitting this is a common tenfold error. Note also that TCID50 counts only particles capable of causing CPE in that cell line; total particle counts by electron microscopy typically exceed infectious titre by 100 to 1,000-fold, since most virions are defective or fail to establish infection.

Worked Examples

Example 1: SARS-CoV-2: 50% CPE at 10^-5.5 dilution, 0.1mL/well

Titer=10^5.5/0.1

Result: Titer=3.16×10⁶ TCID50/mL

Moderate virus stock

Example 2: Reed-Muench: 80% CPE at 10^-5, 20% at 10^-6

Interp: (80-50)/(80-20)=0.5 steps

Result: 50% dilution at 10^-5.5

Standard interpolation method

Example 3: The volume correction

Endpoint at 10^−5.5, 0.1 mL per well

Result: 3.16 × 10⁶ TCID50/mL

The endpoint gives 10^5.5 per 0.1 mL. Dividing by 0.1 converts to per-mL units.

Example 4: Particle-to-PFU ratio

Electron microscopy count versus infectious titre

Result: 100 to 1,000-fold higher

Most virions fail to establish infection. Infectivity assays measure functional particles, not total virus.

Common Mistakes

⚠️

Forgetting the volume correction

The endpoint gives TCID50 per inoculum volume. With 0.1 mL per well, titre per mL is ten times higher — omitting this is a tenfold error.

⚠️

Treating TCID50 and PFU as interchangeable

PFU is roughly 0.7 times TCID50 by Poisson statistics, but the relationship is virus-specific and should be validated rather than assumed.

⚠️

Assuming infectious titre reflects particle count

Total particles typically exceed infectious units by 100 to 1,000-fold, since many virions are defective.

⚠️

Reading wells too early or too late

CPE develops over days and varies by virus. Scoring before it is complete underestimates titre; overgrowth of controls makes late reading unreliable.

Frequently Asked Questions

TCID50 vs PFU?

TCID50 (tissue culture infectious dose 50%): dilution at which 50% of wells show CPE. PFU (plaque forming unit): visible plaques in agar overlay. Both measure infectious virus. PFU ≈ 0.7×TCID50 (statistically: Poisson). PFU more precise but requires plaque assay.

Reed-Muench vs Spearman-Karber?

Reed-Muench: uses cumulative data, simpler. Spearman-Karber: uses proportions at each dilution, more statistically rigorous. Both give similar results. Karber formula: log TCID50 = last dilution - d(Σp - 0.5) where d=log dilution step, p=fraction positive.

What is the difference between TCID50 and PFU?

TCID50 is a statistical endpoint — the dilution infecting half the wells. PFU counts discrete plaques. PFU is roughly 0.7 times TCID50.

Which endpoint method should I use?

Spearman–Kärber is generally preferred because it provides a variance estimate and therefore confidence intervals. Reed–Muench gives only a point estimate.

Why divide by the inoculum volume?

Because the endpoint dilution gives TCID50 per volume inoculated. Converting to per-mL requires dividing by that volume.

Why is infectious titre so much lower than particle count?

Most virions are defective or fail to establish infection. Particle-to-infectivity ratios of 100 to 1,000 are typical.