Last reviewed: October 2026 by Dr. Farrukh Mehmood, Ph.D., 16+ years in pharmaceutical QC/QA
Key takeaways
- Serial dilution is a stepwise dilution where each tube is diluted by the same factor (usually 10-fold), so concentration falls 10−1, 10−2, 10−3…
- Dilution factor of one step = sample ÷ (sample + diluent). 1 mL into 9 mL = 1/10.
- Total dilution = multiply the step dilutions: 1/10 × 1/10 × 1/10 = 1/1000 (10−3).
- CFU/mL = colonies ÷ (volume plated in mL × dilution of that tube).
- In pharma QC (USP <61>), count the plates with the highest number of colonies below 250 (TAMC) or 50 (TYMC).
Dilution is the process of making a solution weaker or less concentrated. In microbiology, serial dilutions (also called log or tenfold dilutions) bring a dense bacterial suspension down to a concentration that suits the test method, or to a level that is easy to count on an agar plate. This guide explains the serial dilution method step by step, gives the formulas, works through real QC calculations, and ends with a free calculator. Use it as a learning aid alongside, not in place of, your approved laboratory SOPs.
What is a serial dilution?
A serial dilution is a series of dilutions made one after another, where each tube is prepared from the tube before it. Because the dilution factor at each step is usually constant, the concentration falls in a geometric (logarithmic) progression. Starting with 108 CFU/mL and diluting 10-fold six times gives 102 CFU/mL.
Log (tenfold) dilution
A log dilution reduces concentration by a factor of ten. Add 1 mL of sample to 9 mL of diluent to give a 10 mL total. The dilution is 1/10, often written 1:10. For example, a suspension of 1,000 CFU/mL becomes 100 CFU/mL after one log dilution.
Watch the notation: in microbiology, “1:10” normally means 1 part in 10 parts total (1 + 9). In some chemistry texts a ratio of 1:10 means 1 part plus 10 parts (1/11). Always write the volumes in your worksheet to avoid ambiguity.
Multiple dilutions
When you need to drop the concentration by several logs, you repeat the step. To go from 35,000 CFU/mL (about 104) to about 35 CFU/mL you need three tenfold steps (10−3), because 35,000 × 1/1000 = 35.
Dilution factor table
The same dilution can be made with different volumes. Use this table to pick the combination that suits your pipettes and tubes.
| Transfer (sample) | Diluent | Total volume | Step dilution | Dilution factor |
|---|---|---|---|---|
| 1 mL | 1 mL | 2 mL | 1/2 | 2 |
| 0.5 mL | 4.5 mL | 5 mL | 1/10 (10−1) | 10 |
| 1 mL | 9 mL | 10 mL | 1/10 (10−1) | 10 |
| 100 µL | 900 µL | 1 mL | 1/10 (10−1) | 10 |
| 10 g or 10 mL product | 90 mL | 100 mL | 1/10 (10−1) | 10 |
| 1 mL | 99 mL | 100 mL | 1/100 (10−2) | 100 |
| 10 µL | 990 µL | 1 mL | 1/100 (10−2) | 100 |
| 0.1 mL | 9.9 mL | 10 mL | 1/100 (10−2) | 100 |
Purpose of serial dilution
- Counting viable organisms: a dense culture or product sample is diluted until a plate gives a countable number of colonies, so the original CFU/mL or CFU/g can be calculated.
- Preparing test inocula: growth promotion tests, method suitability and preservative efficacy tests need suspensions with a defined number of organisms, for example not more than 100 CFU.
- Overcoming antimicrobial activity: diluting a product reduces the effect of preservatives or active ingredients so organisms can recover.
- Bioassays and MIC work: twofold dilutions of an antibiotic or test substance give a concentration series.
Requirements
- Sterile diluent: buffered sodium chloride–peptone solution pH 7.0, phosphate buffer pH 7.2, or 0.9% saline as specified by your method
- Sterile screw-cap tubes or bottles, pre-filled with the exact diluent volume
- Calibrated micropipettes and sterile filtered tips (or sterile graduated pipettes)
- Vortex mixer
- Culture media: Soybean-Casein Digest Agar (TSA) for bacteria and Sabouraud Dextrose Agar (SDA) for yeasts and moulds; selective media as required
- Sterile Petri dishes, spreader (for spread plates), and water bath at about 45 °C (for pour plates)
- Incubators at the temperatures required by the method
- Biosafety cabinet or laminar air flow unit
Serial dilution procedure, step by step
- Disinfect the work area and work aseptically in a biosafety cabinet or LAF.
- Label the tubes 10−1, 10−2, 10−3 and so on, and label the plates with the dilution and replicate number.
- Dispense 9 mL of sterile diluent into each tube (or use pre-filled tubes and verify the volume).
- Mix the sample or culture well. Transfer 1 mL into the first tube. This is the 10−1 dilution.
- Vortex the tube for 5–10 seconds so organisms are evenly distributed.
- With a fresh pipette tip, transfer 1 mL from the 10−1 tube into the next tube to make 10−2. Mix again.
- Repeat until you reach the last dilution you need. You do not need ten tubes; choose enough steps to bring the expected count into the countable range.
- Plate the chosen dilutions in duplicate: 1 mL by the pour-plate method (add about 15–20 mL of molten agar cooled to about 45 °C and swirl), or 0.1 mL spread over the surface of a pre-poured plate.
- Include a negative control: plate the diluent alone to confirm it is sterile.
- Let the agar set, invert the plates and incubate. For pharmaceutical TAMC/TYMC testing per USP <61>: TSA at 30–35 °C for 3–5 days, SDA at 20–25 °C for 5–7 days. For general bacteriology, follow your method (often 35 ± 2 °C for 24–48 h).
Reading the results: which plate do you count?
Only plates in the countable range give a reliable result. Too many colonies merge and are under-counted (TNTC, too numerous to count). Too few colonies give a statistically weak estimate.
- General microbiology: 30–300 colonies per plate. The FDA Bacteriological Analytical Manual (BAM) uses 25–250.
- Pharmaceutical QC, USP <61>: use the dilution with the highest number of colonies below 250 for TAMC (bacteria on TSA) and below 50 for TYMC (yeasts and moulds on SDA).
- Take the arithmetic mean of duplicate plates before calculating.
Serial dilution formulas
1. Dilution of one tube
Dilution = sample volume ÷ (sample volume + diluent volume)
1 mL into 9 mL → 1 ÷ (1 + 9) = 1/10
2. Total (serial) dilution
Total dilution = step 1 × step 2 × step 3 …
1/10 × 1/10 × 1/100 = 1/10,000 (10−4)
3. Organisms in the original sample
CFU/mL (or CFU/g) = colonies counted ÷ (volume plated in mL × dilution)
52 colonies from 0.1 mL of 10−6 → 52 ÷ (0.1 × 10−6) = 5.2 × 108 CFU/mL
4. Number of tenfold steps needed
Steps = log10(starting concentration ÷ target concentration)
Worked examples
Example 1: Counting a bacterial culture
You dilute a stock culture using a mix of 10 µL into 990 µL steps (1/100) and 100 µL into 900 µL steps (1/10). Then you plate:
| Plate | Volume plated | Tube dilution | Colonies | Use it? |
|---|---|---|---|---|
| A | 10 µL | 10−3 | TNTC | No, too many |
| B | 100 µL | 10−5 | 685 | No, above 300 |
| C | 100 µL | 10−6 | 52 | Yes |
| D | 100 µL | 10−7 | 4 | No, below 30 |
Only plate C is in the 30–300 range. CFU/mL = 52 ÷ (0.1 mL × 10−6) = 5.2 × 108 CFU/mL in the original culture. As a check, plate D predicts about 5 colonies (52 ÷ 10), and it shows 4. Plate B predicts about 520 and shows 685. Both agree within normal plating variability.
Example 2: TAMC of a non-sterile tablet (pharma QC)
10 g of crushed tablets is suspended in 90 mL of buffered sodium chloride–peptone solution, giving a 10−1 dilution. A further 10−2 dilution is made (1 mL + 9 mL). 1 mL of each dilution is pour-plated in duplicate on TSA and incubated at 30–35 °C for 5 days.
- 10−1 plates: 48 and 52 colonies. Mean = 50.
- 10−2 plates: 6 and 4 colonies. Mean = 5.
The 10−1 plates have the highest count below 250, so they are used. TAMC = 50 ÷ (1 mL × 10−1) = 500 CFU/g. For a non-aqueous oral preparation, USP <1111> sets the TAMC criterion at 103 CFU/g, interpreted as a maximum acceptable count of 2,000. This batch complies.
Example 3: Preparing an inoculum of not more than 100 CFU
For a growth promotion test you need not more than 100 CFU per inoculum. A 0.5 McFarland suspension of E. coli contains roughly 1.5 × 108 CFU/mL.
- Steps needed to reach about 150 CFU/mL: log10(1.5 × 108 ÷ 150) = 6. So you make six tenfold dilutions, to 10−6.
- The 10−6 tube holds about 150 CFU/mL. Inoculating 0.5 mL gives about 75 CFU, within the limit.
- McFarland values are only estimates, so always confirm the actual count by plating the same volume of the inoculum in parallel.
Example 4: How many dilutions do I need?
You expect about 2 × 107 CFU/mL and want about 100 colonies from a 1 mL pour plate. Steps = log10(2 × 107 ÷ 100) = log10(2 × 105) ≈ 5.3. Plate the 10−5 and 10−6 dilutions, since about 200 and 20 colonies are expected, which brackets the target.
Serial dilution and CFU calculator
Use these calculators to check your worksheet. They do not replace the calculation and review steps in your SOP.
Tip: write numbers like 1.5e8 for 1.5 × 108.
Common mistakes and how to avoid them
- Reusing the pipette tip: organisms clinging to the tip are carried into the next tube and inflate later counts. Use a fresh tip for every transfer.
- Poor mixing: vortex every tube before taking the next aliquot. Settled cells give uneven results.
- Forgetting the plated volume: 0.1 mL spread plates need an extra ×10 in the calculation compared with 1 mL pour plates.
- Wrong diluent volume: autoclaving or evaporation can reduce pre-filled volumes. Verify 9.0 mL before use.
- Molten agar too hot: agar above about 45–48 °C can injure organisms in pour plates.
- Counting out-of-range plates: report TNTC or “less than” values according to your SOP instead of forcing a calculation from a crowded or nearly empty plate.
- Ignoring product inhibition: if the product is antimicrobial, prove recovery during method suitability (USP <61>/<62>) using dilution, neutralizers or membrane filtration.
Frequently asked questions
What is the formula for serial dilution?
Each step’s dilution is sample ÷ (sample + diluent). The total dilution is the product of all step dilutions. The original concentration is colonies ÷ (volume plated × total dilution).
What is the difference between dilution and dilution factor?
The dilution is the fraction of the original sample in the tube, for example 1/10 or 10−1. The dilution factor is its reciprocal, 10. Multiplying the colony count by the dilution factor (and correcting for plated volume) gives CFU/mL.
Why are tenfold dilutions used?
Tenfold steps are easy to calculate (each tube is one log lower) and they cover a wide range of concentrations with few tubes, so at least one plate usually falls in the countable range.
How many colonies should a countable plate have?
Usually 30–300 (25–250 in FDA BAM). For pharmacopoeial microbial enumeration under USP <61>, use the plates with the highest count below 250 for TAMC and below 50 for TYMC.
Pour plate or spread plate: which volume should I use?
Pour plates usually take 1 mL of the dilution mixed with molten agar. Spread plates take 0.1 mL spread on the surface. Use the actual volume in the CFU formula.
Test yourself: try our free QC microbiology quiz, or the broader microbiology quiz.
Related guides
- Growth promotion test of culture media
- Total microbial count (TAMC and TYMC)
- Minimum inhibitory concentration (MIC) guide
- OOS procedure in pharma
References
- USP General Chapter <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests.
- USP General Chapter <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms.
- USP General Chapter <1111> Microbiological Examination of Nonsterile Products: Acceptance Criteria for Pharmaceutical Preparations and Substances for Pharmaceutical Use.
- FDA Bacteriological Analytical Manual, Chapter 3: Aerobic Plate Count.
Part of our QC microbiology guide, which maps every microbiology test, guide and tool on AskPharmaTutor in the order a lab uses them.
