Free TCID50 Calculator (Spearman-Kärber and Reed-Muench)

Enter the number of infected wells at each dilution and get your titer in TCID50/mL straight away. The calculator works out the Spearman-Kärber and Reed-Muench results side by side, gives a 95% confidence interval, estimates PFU/mL, and warns you when the plate itself is the problem. It runs in your browser, and nothing you enter is sent to TotalLab.

TCID50 calculator

How to use the TCID50 calculator

1. Enter the first dilution you plated as “1 in …”. For a series that starts at 10^-1, enter 10.
2. Enter the dilution factor between wells (10 for tenfold, 4 for fourfold, 2 for twofold).
3. Enter the number of dilutions and the number of replicate wells per dilution.
4. Enter the inoculum volume per well in mL (100 µL is 0.1).
5. For each dilution, enter how many wells showed cytopathic effect (CPE) or scored positive. If a well was lost, lower the “wells tested” number for that dilution.
6. Select Calculate titer.

The result is the titer of the sample you added to the first dilution, in TCID50/mL. If you prediluted the stock before the plate, multiply the result by that predilution.

How the calculation works

Both methods estimate the dilution at which half of the inoculated wells would be infected. Both were introduced nearly a century ago and remain the most commonly used methods [1].

Spearman-Kärber
log10 endpoint = x0 − d/2 + d × Σp

x0 is the log10 of the reciprocal of the highest dilution at which every well is infected. d is the log10 of the dilution factor (1 for tenfold). Σp is the sum of the proportion of infected wells at x0 and at every higher dilution. The standard error is d × √Σ[p(1 − p)/(n − 1)], where n is the number of wells at each dilution, and the 95% confidence interval is the estimate ± 1.96 × SE [2].

Reed-Muench
Reed-Muench adds up infected wells from the most dilute well back toward the most concentrated, and uninfected wells in the other direction. It then finds the two dilutions whose cumulative percent infected sit either side of 50% and interpolates between them:

proportionate distance = (% infected above 50% − 50) / (% infected above 50% − % infected below 50%)
log10 endpoint = log10 of the dilution above 50% + proportionate distance × d

Finally, the endpoint is converted to TCID50/mL by dividing by the inoculum volume in mL.

On a clean plate, like the worked example below, the two results agree closely. They drift apart when infection does not fall steadily with dilution, which is why the calculator flags that case.

Worked example

Select Load example in the calculator to see this plate. Eight wells per dilution, tenfold dilutions from 1:10, 0.1 mL per well. Infected wells: 8, 8, 8, 8, 6, 3, 1, 0. Reading the table: every well is infected down to 1:10,000, the proportion infected then falls through 0.750, 0.375 and 0.125, and no well is infected at 1:100,000,000. The 50% endpoint therefore sits between the 1:100,000 and 1:1,000,000 dilutions, which is the point both methods interpolate. The cumulative columns are the running totals Reed-Muench uses to find it.

Example endpoint dilution plate and the Reed-Muench cumulative values.

Spearman-Kärber: the highest dilution with every well infected is 1:10,000, so x0 = 4. Σp from there is 1 + 0.75 + 0.375 + 0.125 + 0 = 2.25. The endpoint is 4 − 0.5 + 2.25 = 5.75, which is 10^5.75 TCID50 per 0.1 mL, or 10^6.75 = 5.6 × 10^6 TCID50/mL. The 95% confidence interval is ± 0.54 log10. Reed-Muench: the cumulative percent infected is 83.3% at 1:10^5 and 36.4% at 1:10^6. The proportionate distance is (83.3 − 50) / (83.3 − 36.4) = 0.71, so the endpoint is 5.71, or 10^6.71 = 5.1 × 10^6 TCID50/mL.

Converting TCID50 to PFU

If a virus sample is diluted to one TCID50 per well, the Poisson distribution predicts about ln 2 = 0.69 infectious units per well. That is where the familiar rule of thumb comes from: PFU/mL ≈ 0.7 × TCID50/mL [1]. The calculator uses 0.69.

Treat the converted number as an estimate. The same authors showed that Reed-Muench and Spearman-Kärber themselves overestimate the TCID50, and suggest multiplying by 0.561 instead of 0.69 when you need an accurate multiplicity of infection [1]. A plaque assay on the same cells remains the direct measure.

Getting a better TCID50 result

The calculator can only be as good as the plate. Four habits help.

Start concentrated enough that the first dilutions are fully infected, and dilute far enough that the last are fully clear. Both methods lose accuracy as the 50% point approaches either end of the series [1].

Prefer more dilutions with a smaller dilution factor over more replicates of fewer dilutions. With the same number of wells, closer dilutions give a finer and more accurate estimate [1].

Check that infection falls steadily with dilution. A dilution with more infected wells than the one before it usually means a pipetting or scoring problem.

Use a checked tool. As Cresta and colleagues put it, “Many research groups rely on spreadsheet calculators that are passed down through generations of trainees or found on the internet, and can contain errors” [1].

When the endpoint assay is the bottleneck

A TCID50 plate is read once, at the end, after several days of incubation. For a lab releasing viral vectors or oncolytic viruses, that is the slow step.

TiterKinetix from TotalLab takes a different route. It analyzes the kinetic infectious virus titer (KIT) assay, which follows infection-induced changes in cell morphology in bright-field images over time instead of scoring a single endpoint [3]. Titers are calculated against a reference standard.

• Results in 24 hours instead of a 5 to 10 day TCID50
• CV under 20% from three wells per sample
• About 400 samples per operator per week
• Label-free: no stains or specialist reagents
• Runs on the plate imagers many labs already have, including the Agilent BioTek Cytation 5 and the Sartorius Incucyte, in 96- and 384-well formats
• Audit trail, electronic signatures and user access control through AuditSafe, for 21 CFR Part 11 and GMP work

See how TiterKinetix works

Frequently Asked Questions

What is TCID50?

TCID50 is the 50% tissue culture infectious dose: the amount of virus that infects half of the inoculated cell cultures. A titer in TCID50/mL is the number of those doses in one mL of the sample.

Which is better, Spearman-Kärber or Reed-Muench?

Spearman-Kärber uses every dilution and gives a standard error, so it is the more common choice for reporting. Reed-Muench is widely used and usually agrees closely. Both are approximations; published simulations show both carry a bias, which grows when the 50% point is near the edge of the plate [1].

How do I calculate the 95% confidence interval for a TCID50?

For Spearman-Kärber, the standard error of the log10 endpoint is d × √Σ[p(1 − p)/(n − 1)]. The 95% confidence interval is the log10 titer ± 1.96 × SE [2]. The calculator does this for you when there are at least two wells per dilution.

How do I convert TCID50 to PFU?

Multiply TCID50/mL by about 0.69 (ln 2) to estimate PFU/mL. Because Reed-Muench and Spearman-Kärber overestimate the TCID50, a factor of 0.561 gives a closer estimate of infectious units [1].

My first dilution is not 100% infected. Can I still calculate a titer?

The calculator will give a result and a warning. The estimate is less reliable because the plate did not capture the full transition. Repeat with a more concentrated starting dilution.

Does the calculator work with twofold or fourfold dilutions?

Yes. Enter any dilution factor greater than 1.

Is my data stored?

No. The calculation runs in your browser and nothing is sent to TotalLab.

Is there a faster alternative to the TCID50 assay?

The kinetic infectious virus titer (KIT) assay reads infection-induced cell rounding over time and gives titers in 24 hours [3]. TiterKinetix from TotalLab automates the analysis on imagers such as the Cytation 5 and Incucyte.

References

[1] Cresta D, Warren DC, Quirouette C, Smith AP, Lane LC, Smith AM, Beauchemin CAA. Time to revisit the endpoint dilution assay and to replace the TCID50 as a measure of a virus sample’s infection concentration. PLoS Comput Biol. 2021;17(10):e1009480. doi:10.1371/journal.pcbi.1009480
(Source for: Spearman-Kärber and Reed-Muench as the standard methods; spreadsheet-calculator quotation; 0.69 and 0.561 conversion factors; bias near the plate limits; dilutions versus replicates)

[2] Hamilton MA, Russo RC, Thurston RV. Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environ Sci Technol. 1977;11(7):714-719. doi:10.1021/es60130a004
(Source for: Spearman-Kärber standard error formula)

[3] Hotter D, Kunzelmann M, Kiefer F, Leukhardt C, Fackler C, Jäger S, Solzin J. High-Throughput Determination of Infectious Virus Titers by Kinetic Measurement of Infection-Induced Changes in Cell Morphology. Int J Mol Sci. 2024;25(15):8076. doi:10.3390/ijms25158076
(Source for: KIT assay principle, results within 24 h, CV < 20% from three wells, ~400 samples per operator per week)

[4] Ramakrishnan MA. Determination of 50% endpoint titer using a simple formula. World J Virol. 2016;5(2):85-86. doi:10.5501/wjv.v5.i2.85
(Source for: further reading on Spearman-Kärber and Reed-Muench)

[5] TotalLab. TiterKinetix product page. https://totallab.com/titerkinetix-kinetic-virus-infectivity-analysis-software/
(Source for: all TiterKinetix claims: 5 to 10 day comparison, instruments, well formats, AuditSafe)

Still waiting days for a TCID50?

TiterKinetix reads infectious titer from kinetic bright-field images in 24 hours, on the imager you already have. See it on your own virus-cell system.

Talk to us: +44 191 255 8899 or info@totallab.com