Difficulty: Beginner to Intermediate | Prerequisites: Lab Safety and Micropipette Technique (BIO Lab 01, Part 1). You need to be comfortable pipetting accurate volumes before this material will make sense in practice.
Serial dilutions are one of the most common procedures in biology, used whenever you need to reduce a solution's concentration in a controlled, stepwise fashion. This lab teaches you how to perform a 1:10 serial dilution series and how to calculate concentrations using the dilution equation C1V1 = C2V2. These skills come up repeatedly in microbiology (plating bacteria at countable densities), pharmacology (testing drugs across a concentration range), and molecular biology (setting up standard curves). If you can set up a dilution series and solve for any variable in C1V1 = C2V2, you have the quantitative foundation for most wet-lab work in biology.
A serial dilution takes a small portion of one solution and mixes it into a larger volume of solvent, repeating the process to get progressively weaker concentrations. The dilution factor at each step multiplies across steps (e.g. three rounds of 1:10 = 1:1,000 overall). To find any unknown concentration or volume, use C1V1 = C2V2 and solve for the missing variable.
Dilution
The process of reducing the concentration of a solute in a solution by adding more solvent. Think of it as watering something down in a precise, measured way.
Dilution factor
The ratio of the volume of sample transferred to the total volume of the new solution. For example, transferring 100 µL into 900 µL of water gives a total of 1,000 µL and a dilution factor of 1/10 (or 1:10). In simple terms, it tells you what fraction of the original strength you end up with after one dilution step.
Serial dilution
A series of stepwise dilutions where each new tube is diluted from the previous one, not from the original stock. Each step multiplies the dilution factor. In simple terms, you dilute, then dilute the dilution, then dilute that, and so on.
Stock solution
The original, undiluted solution at its starting concentration. This is what you begin with before any dilution steps.
C1V1 = C2V2 (the dilution equation)
The equation that relates concentration and volume before and after a dilution. C1 is the starting concentration, V1 is the volume you take from the starting solution, C2 is the final concentration, and V2 is the final total volume. Think of it as "what you start with equals what you end up with" in terms of the total amount of solute.
Concentration
The amount of solute per unit volume of solution, often expressed as mg/mL, µg/mL, or units/mL. In simple terms, how strong or weak the solution is.
Solvent
The liquid (usually water in biology labs) used to dilute the solute. It is what you add to bring the volume up.
1:10 dilution
One part sample to nine parts solvent, giving a total of ten parts. The sample's concentration drops to one-tenth of what it was. This is the standard dilution step used in this lab.
The procedure (1:10 series from this lab)
Start with six labelled microcentrifuge tubes per colour (e.g. R1, O1, Y1, G1, B1, V1 for series 1).
Add 900 µL of water to each tube using the P-1000.
Add 100 µL of concentrated dye to the first tube (e.g. R1) using the P-200. Cap, invert, and flick to mix.
Transfer 100 µL from R1 into R2 (which already contains 900 µL of water). Mix. This is your first 1:10 dilution.
Repeat: transfer 100 µL from R2 into R3, mix. Continue through the series.
Each transfer is 100 µL into a total of 1,000 µL (100 µL sample + 900 µL water), so the dilution factor at each step is 100/1,000 = 1/10.
What you observe
The colour intensity decreases with each tube. Series 1 is the darkest, series 2 is lighter, and series 3 is lighter still.
This is a direct visual demonstration of concentration dropping by a factor of 10 at each step.
Calculating overall dilution
Multiply the dilution factor at each step.
After two 1:10 steps: 1/10 x 1/10 = 1/100.
After three 1:10 steps: 1/10 x 1/10 x 1/10 = 1/1,000.
If your starting concentration is 1 unit/mL, after three rounds of 1:10 dilution the concentration is 0.001 unit/mL.
Example from the lab: six tubes (Stock, A, B, C, D, E, Final)
Each tube contains 400 µL, and 80 µL is transferred at each step.
Dilution factor per step = volume transferred / total volume = 80 / (80 + 400) = 80/480 = 1/6. (Note: some versions of this lab use 80 µL into 400 µL already present, giving a total of 480 µL per tube. Check your own lab sheet for the exact volumes.)
However, looking at the student answers from the lab: the dilution factor per step is written as 1/5. This means 80 µL was transferred into 320 µL of solvent (total 400 µL), giving 80/400 = 1/5.
Dilution from Stock to Tube C (three steps): 1/5 x 1/5 x 1/5 = 1/125 = 0.008.
Dilution from Stock to Tube B (two steps): 1/5 x 1/5 = 1/25 = 0.04.
Dilution factor for the final tube (one step from E): 1/5.
This equation lets you solve for any one unknown when you know the other three values. The key insight: the total amount of solute is conserved. What you take out of the concentrated solution ends up in the dilute solution.
Worked Example 1: How much stock to add?
Given: C1 = 2 mg/mL (stock), C2 = 500 µg/mL = 0.5 mg/mL (desired), V2 = 1 mL (final volume).
Solve for V1: V1 = (C2 x V2) / C1 = (0.5 mg/mL x 1 mL) / 2 mg/mL = 0.25 mL = 250 µL.
You would add 250 µL of stock solution, then top up to 1 mL total with water (add 750 µL water).
Worked Example 2: Concentration of a new TAE buffer
Given: C1 = 50 mg/mL (stock TAE), V1 = 100 mL, V2 = 4,000 mL (4 L final volume).
Solve for C2: C2 = (C1 x V1) / V2 = (50 mg/mL x 100 mL) / 4,000 mL = 1.25 mg/mL.
Worked Example 3: What final volume do I need?
Given: C1 = 10 mg/mL (stock buffer), V1 = 5 mL, C2 = 50 µg/mL = 0.05 mg/mL.
Solve for V2: V2 = (C1 x V1) / C2 = (10 mg/mL x 5 mL) / 0.05 mg/mL = 1,000 mL.
Worked Example 4: Finding the stock concentration
Given: V1 = 10 µL (volume of stock added), C2 = 75 µg/mL (working solution concentration), V2 = 500 µL (final volume).
Solve for C1: C1 = (C2 x V2) / V1 = (75 µg/mL x 500 µL) / 10 µL = 3,750 µg/mL = 3.75 mg/mL.
Unit conversion: 3,750 µg/mL ÷ 1,000 = 3.75 mg/mL.
The dilution equation
C1 x V1 = C2 x V2
Where C1 = initial concentration, V1 = initial volume (the amount you transfer), C2 = final concentration, V2 = final total volume.
Rearranged to solve for each variable:
V1 = (C2 x V2) / C1
C2 = (C1 x V1) / V2
V2 = (C1 x V1) / C2
C1 = (C2 x V2) / V1
Overall dilution factor for a serial dilution
Overall dilution = (dilution factor per step)^n, where n = number of steps.
For a 1:10 series with 3 steps: (1/10)^3 = 1/1,000.
Dilution factor per step
Dilution factor = volume transferred / total volume in the new tube.
Example: 100 µL transferred into 900 µL water = 100/1,000 = 1/10.
Unit conversions to watch for
1 mg = 1,000 µg. 1 mL = 1,000 µL. Always convert C1 and C2 to the same units before plugging into the equation.
Serial dilutions are how microbiologists count bacteria: you dilute a sample until individual colonies are countable on a plate, then back-calculate the original concentration. Pharmacologists use dilution series to test drugs across a range of doses in a single experiment. Clinical labs dilute patient samples to bring analyte concentrations into the measurable range of their instruments. The C1V1 = C2V2 equation is used daily by anyone preparing buffers, staining solutions, or reagents from concentrated stocks.
Students often confuse V2 (the final total volume) with the volume of solvent added. V2 is the total volume in the tube after mixing, not just the water you poured in. If you add 100 µL of sample to 900 µL of water, V2 is 1,000 µL, not 900 µL.
Students sometimes think a 1:10 dilution means dividing by 10 at each step but forget to multiply the dilution factors together across steps. Three rounds of 1:10 is 1/1,000, not 1/30.
Students frequently forget to convert units before using C1V1 = C2V2. If C1 is in mg/mL and C2 is in µg/mL, you must convert one to match the other before solving. The equation only works when both concentrations use the same units.
Students sometimes think "dilution factor" and "concentration" are the same thing. The dilution factor is the fraction by which you reduced the concentration. The concentration is the result after applying that factor to the original value.
⚠️ Be able to calculate the overall dilution from a series of steps by multiplying dilution factors together.
⚠️ Know how to set up and solve C1V1 = C2V2 for any of the four variables. Expect at least one problem where you solve for V1, one for C2, and possibly one for C1.
⚠️ Watch your unit conversions. A common exam trap gives you C1 in mg/mL and C2 in µg/mL. Convert before you calculate.
⚠️ Be able to identify the dilution factor from a diagram showing tubes with labelled transfer volumes and total volumes.
⚠️ Understand the difference between the dilution factor at a single step and the cumulative dilution across multiple steps.
True or False: In a 1:10 dilution, you add 1 part sample to 10 parts solvent. (False. You add 1 part sample to 9 parts solvent, for a total of 10 parts.)
Fill in the blank: After four rounds of 1:10 serial dilution, the overall dilution factor is ______. (1/10,000 or 10^-4)
True or False: In C1V1 = C2V2, V2 refers to the volume of water you add. (False. V2 is the total final volume, including the sample.)
Fill in the blank: 2,500 µg/mL = ______ mg/mL. (2.5 mg/mL)
True or False: If you transfer 200 µL of a solution into 800 µL of water, the dilution factor is 1/4. (False. The dilution factor is 200/1,000 = 1/5.)
Q: You perform a serial dilution with a 1:5 dilution factor at each step. After 3 steps, what is the overall dilution?
A: (1/5)^3 = 1/125. If your stock was 1 mg/mL, the concentration after 3 steps is 1/125 mg/mL = 0.008 mg/mL.
Q: You have a stock solution at 10 mg/mL. You need 500 µL of a 0.2 mg/mL working solution. How much stock do you add, and how much water?
A: C1V1 = C2V2. V1 = (C2 x V2) / C1 = (0.2 mg/mL x 500 µL) / 10 mg/mL = 10 µL. Add 10 µL of stock, then add 490 µL of water to reach 500 µL total.
Q: You transfer 100 µL from a 1 unit/mL solution into 900 µL of water (tube 1). You then transfer 100 µL from tube 1 into 900 µL of water (tube 2). What is the concentration in tube 2?
A: Each step is a 1:10 dilution. After step 1: 1 x 1/10 = 0.1 unit/mL. After step 2: 0.1 x 1/10 = 0.01 unit/mL.
Q: A protocol tells you to add 10 µL of stock to a tube, then add water to a final volume of 500 µL. The working concentration should be 75 µg/mL. What is the concentration of your stock?
A: C1 = (C2 x V2) / V1 = (75 µg/mL x 500 µL) / 10 µL = 3,750 µg/mL = 3.75 mg/mL.
Q: You are making 4 L of TAE buffer from a 50 mg/mL stock. You add 100 mL of stock and fill to 4 L with water. What is the final concentration?
A: C2 = (C1 x V1) / V2 = (50 mg/mL x 100 mL) / 4,000 mL = 1.25 mg/mL.
Serial dilutions feed directly into microbiology labs where you plate bacteria at different dilutions to count colony-forming units (CFUs). The C1V1 = C2V2 equation is the same principle behind preparing solutions for gel electrophoresis (loading buffers, staining solutions), PCR master mixes, and enzyme assays. Understanding concentration and dilution also connects to pharmacology (dose-response curves) and analytical chemistry (calibration standards).
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