How to Do Serial Dilution (Step-by-Step with Tables & Calculator)

Serial dilution is a core laboratory technique used to reduce the concentration of a solution in a stepwise manner.
It is widely applied in molecular biology, microbiology, pharmacology, biochemistry, and many other research areas.
Whether you are preparing a standard curve for a spectrophotometric assay, reducing bacterial concentration for plating, or adjusting chemical concentrations for a reaction, mastering this method will improve your accuracy.

In this guide, you’ll learn:

• What a serial dilution is
• Step-by-step instructions for preparing eight dilution levels (including stock and blank)
• Tables for both 2-fold and 10-fold dilution series
• How to set this up for tubes or a 96-well plate
• The role of dilution factors
• How to speed up the process using our Serial Dilution Calculator.

What Is Serial Dilution?

Serial dilution is a method of diluting a solution multiple times by the same factor, for example it can be like 1:2 (two-fold) or 1:10 (ten-fold), in a sequence.
Each dilution step uses a measured volume of the solution from the previous step (tube or well), mixing it with a measured volume of diluent (such as water, PBS, or buffer).

This approach helps achieve large overall dilutions without needing to pipette extremely small volumes.
For example:

But a series of three 1:10 dilutions gives the same result (1:10 × 1:10 × 1:10 = 1:1000), and is much more accurate.

A single step dilution of 1:1000 is hard to measure accurately

When to Use Serial Dilutions

Serial dilutions are used in many lab workflows, such as:

• Preparing standard curves for absorbance or fluorescence measurements
• Determining bacterial or viral titers in microbiology
• Adjusting reagent concentrations in enzyme kinetics experiments
• Creating a range of drug concentrations in dose–response assays

Because of its flexibility, the method can be scaled for test tubes, microcentrifuge tubes, or multi-well plates.

Planning a Serial Dilution

Before starting, decide:

1. Your dilution factor (e.g., 1:2 or 1:10 or any other customized)
2. The total number of dilutions (including stock and blank)
3. Final volume in each tube/well (for this guide, we use 1 mL)
4. Type of diluent (must be compatible with your experiment, it can be something like buffer or water)

💡 Tip: For highest accuracy, always use calibrated pipettes or graduated cylinders.

Step-by-Step Example 1:2 Serial Dilution 8 Tubes (Including Stock and Blank)

Preparation (Final volume 1mL):

1. Label your tubes:

• Tube 1 (or Row) = Stock (undiluted sample)
• Tube 2–7 (or Row)= Dilutions D1–D6 (6 serial dilutions)
• Tube 8 (or Row)= Blank (diluent only, no sample) – used for background subtraction.

2. Prepare your diluent (e.g., PBS, water):

• Tubes 2–7: Add 0.5 mL (500 μL) of diluent to each tube.
• Tube 8 (Blank): Add 1 mL (1000 μL) of diluent.
• Tube 1 (Stock): Initially empty or keep original stock aside.

3. Set up your pipette:

• Use a calibrated pipette for accurate volume transfer.
• Important: Always change tips between different samples to avoid contamination.

💡Dilution steps are below table

Tube / Well	Label	Diluent (e.g., PBS)	Sample Transferred	Transferred From	Final Volume	Dilution Factor	Relative Conc. (%)
1	Stock (Undiluted)	0 mL	1 mL of stock	–	1 mL	1 (NONE)	100%
2	D1	0.5 mL	0.5 mL from Tube 1	Tube 1	1 mL	1:2	50%
3	D2	0.5 mL	0.5 mL from Tube 2	Tube 2	1 mL	1:4	25%
4	D3	0.5 mL	0.5 mL from Tube 3	Tube 3	1 mL	1:8	12.5%
5	D4	0.5 mL	0.5 mL from Tube 4	Tube 4	1 mL	1:16	6.25%
6	D5	0.5 mL	0.5 mL from Tube 5	Tube 5	1 mL	1:32	3.125%
7	D6	0.5 mL	0.5 mL from Tube 6	Tube 6	1 mL	1:64	1.5625%
8	Blank (B0)	1 mL	0 mL (no analyte)	–	1 mL	N/A	0% (Background)

Stepwise Dilution:

4. Step 1 – First dilution (Tube 1 → Tube 2):
   • Transfer 0.5 mL (500 μL) from the stock (Tube 1) to Tube 2 (already contains 0.5 mL diluent).
   • Mix thoroughly.
   • Final volume in Tube 2: 0.5 mL (diluent) + 0.5 mL (sample) = 1 mL.
5. Step 2 – Second dilution (Tube 2 → Tube 3):
   • Transfer 0.5 mL from Tube 2 to Tube 3 (already contains 0.5 mL diluent).
   • Mix thoroughly.
   • Final volume in Tube 3: 1 mL (0.5 mL sample + 0.5 mL diluent).
   • Tube 2 now contains 1 mL (after transfer), ready for next dilution.
6. Step 3 – Third dilution (Tube 3 → Tube 4):
   • Transfer 0.5 mL from Tube 3 to Tube 4 (already contains 0.5 mL diluent).
   • Mix thoroughly.
   • Final volume in Tube 4: 1 mL.
7. Step 4 – Fourth dilution (Tube 4 → Tube 5):
   • Transfer 0.5 mL from Tube 4 to Tube 5 (already contains 0.5 mL diluent).
   • Mix thoroughly.
   • Final volume in Tube 5: 1 mL.
8. Step 5 – Fifth dilution (Tube 5 → Tube 6):
   • Transfer 0.5 mL from Tube 5 to Tube 6 (already contains 0.5 mL diluent).
   • Mix thoroughly.
   • Final volume in Tube 6: 1 mL.
9. Step 6 – Sixth dilution (Tube 6 → Tube 7):
   • Transfer 0.5 mL from Tube 6 to Tube 7 (already contains 0.5 mL diluent).
   • Mix thoroughly.
   • Final volume in Tube 7: 1 mL.
10. Step 7 – Blank tube (Tube 8):
    • Tube 8 only contains 1 mL of diluent.
    • No sample is added.
    • Used for background measurement in experiments.

Key Notes / Tips for Accuracy:

• Always mix thoroughly after each transfer to ensure uniform concentration.
• Use fresh tips to avoid cross-contamination.
• The final volume in every tube is 1 mL because each tube already contains 0.5 mL of diluent and receives 0.5 mL of sample.
• Stock tube always retains 1 mL (or more, if starting with extra) for sequential dilutions.
• Dilution factor doubles each step: 1:2 → 1:4 → 1:8 → 1:16 → 1:32 → 1:64.
• Relative concentrations decrease as 100% → 50% → 25% → 12.5% → 6.25% → 3.125% → 1.5625%.

1:10 Serial Dilution Table (1 mL final volume per tube)

Tube / Well	Label	Diluent Volume (e.g., PBS)	Sample Transferred	Transferred From	Final Volume	Dilution Factor	Relative Conc. (%)
Tube 1	Stock (Undiluted)	0 mL	1 mL of stock	–	1 mL	1 (NONE)	100%
Tube 2	D1	0.9 mL	0.1 mL from Tube 1	Tube 1	1 mL	1:10	10%
Tube 3	D2	0.9 mL	0.1 mL from Tube 2	Tube 2	1 mL	1:100	1%
Tube 4	D3	0.9 mL	0.1 mL from Tube 3	Tube 3	1 mL	1:1000	0.1%
Tube 5	D4	0.9 mL	0.1 mL from Tube 4	Tube 4	1 mL	1:10,000	0.01%
Tube 6	D5	0.9 mL	0.1 mL from Tube 5	Tube 5	1 mL	1:100,000	0.001%
Tube 7	D6	0.9 mL	0.1 mL from Tube 6	Tube 6	1 mL	1:1,000,000	0.0001%
Tube 8	Blank (B0)	1 mL	0 mL (no analyte)	–	1 mL	N/A	0% (Background)

Use a Serial Dilution Calculator

To avoid manual errors and save time, you can use our free online dilution calculator:

👉 Try the Serial Dilution Calculator Here – Its free, you can download it as excel file or pdf.

Just enter the dilution factor (e.g., 1:2 or 1:10), number of steps, and desired final volume — it will auto-generate the values and instructions for you.

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