What is a serial dilution?

A serial dilution is a stepwise series where each tube is diluted by the same factor from the previous one. Repeating a 1:10 dilution five times gives concentrations spanning five orders of magnitude, which is why the technique is used to count bacteria and titre antibodies.

How is the concentration at each step calculated?

The concentration after step n equals the stock concentration divided by the dilution factor raised to the power n. So a 1000 unit stock at a 1:10 dilution gives 100, 10, 1, and so on. The stock itself is step 0.

How much do I transfer at each step?

The transfer volume is the total tube volume divided by the dilution factor. For a 1:10 series with 1 mL tubes you move 0.1 mL forward into 0.9 mL of fresh diluent. Mix well before the next transfer.

Why change pipette tips between steps?

Residual liquid clinging to a reused tip carries concentrated sample forward and inflates the lower dilutions, which corrupts colony counts. Always use a fresh tip and vortex or pipette-mix each tube before the next transfer.

Can I use this for CFU plate counts?

Yes. Pick the tube whose plated dilution lands in the 30 to 300 colony range, multiply the count by the cumulative dilution factor for that tube, and divide by the plated volume to get CFU per mL of the original sample.

What is the Serial Dilution Calculator?

Compute the concentration at each tube of a serial dilution from a stock concentration, dilution factor, and step count, plus the transfer and diluent volume per step. For microbiology, immunology, and cell biology. It runs free in your browser on Gera Tools, with nothing uploaded.

Serial Dilution Calculator

Name: Serial Dilution Calculator
Creator: Gera Tools
License: https://creativecommons.org/licenses/by/4.0/

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A serial dilution spreads a single stock across many tubes, each diluted by the same factor from the one before it. This calculator shows the concentration at every tube, the cumulative dilution factor, and the exact transfer and diluent volumes so your series is reproducible.

How it works

Each step divides the previous concentration by the same dilution factor, so the concentrations fall geometrically:

concentration[n] = stock / DF^n        (n = 0 is the undiluted stock)
cumulative DF[n]  = DF^n
transfer volume   = total volume / DF
diluent volume    = total volume − transfer volume

A 1:10 (DF = 10) series therefore produces concentrations of stock, stock/10, stock/100, stock/1000, and so on. For 1 mL tubes you move 0.1 mL forward into 0.9 mL of fresh diluent at every step.

Worked example

Starting from a 1,000 µg/mL stock with a dilution factor of 10 over five steps in 1 mL tubes:

Tube	Concentration	Cumulative DF	Transfer	Diluent
Stock (0)	1,000 µg/mL	1×	—	—
1	100 µg/mL	10×	0.1 mL	0.9 mL
2	10 µg/mL	100×	0.1 mL	0.9 mL
3	1 µg/mL	1,000×	0.1 mL	0.9 mL
4	0.1 µg/mL	10,000×	0.1 mL	0.9 mL
5	0.01 µg/mL	100,000×	0.1 mL	0.9 mL

Counting CFU from plated dilutions

For bacterial plate counts, the goal is to find the tube whose plated dilution lands in the 30–300 colony countable range. From that plate:

CFU/mL of original stock = colony count / (volume plated × cumulative DF)

For example, if 0.1 mL from Tube 4 (cumulative DF = 10,000) gives 85 colonies:

CFU/mL = 85 / (0.1 mL × 10,000) = 85,000 CFU/mL

Only count plates in the 30–300 range; fewer than 30 colonies gives statistically unreliable counts, and more than 300 become too crowded to distinguish.

Common sources of error

Reused pipette tips: residual liquid on a tip carries concentrated sample into the next tube, inflating that dilution. This is the most common cause of curved calibration lines that should be linear, and of over-counted plates. Always use a fresh tip and vortex or pipette-mix each tube before the next transfer.

Incomplete mixing: transfer 0.1 mL into 0.9 mL and mix thoroughly before drawing the next transfer. Pulling from an unmixed tube takes sample from whatever concentration zone the pipette tip enters.

Wrong diluent volume: if your total tube volume is not what you entered in the calculator, the transfer fraction changes. For a 2 mL tube with DF = 10, transfer is 0.2 mL into 1.8 mL, not 0.1 mL into 0.9 mL.

Losing track of the stock: always label Tube 0 as the undiluted stock so the numbering stays unambiguous when you come back to the rack 30 minutes later.

Tips

Mix each tube thoroughly before every transfer.
Use a fresh pipette tip per step — carryover is the most common source of error.
For dilutions spanning more than 6 orders of magnitude, consider preparing two overlapping series from the stock and the intermediate tube to reduce cumulative pipetting error.