Serial Dilution Calculator and 96-Well Plate Planner
Plan serial dilutions with exact two-step routes, pipette limits, excess and dead volume. Map replicates on a 96-well plate and back-calculate CFU/mL.
This serial dilution calculator turns a dilution series into a practical tube recipe and a 96-well plate dilution layout. It is intended for students, laboratory engineers, and researchers preparing assay standards or checking viable-count arithmetic. It combines arbitrary dilution factors with replicate demand, dispensing excess, dead volume, and a separate CFU back-calculation.
Choose a repeated factor for a regular series, enter a different factor at every step, or select a two-step target. A factor of 10 means one part of sample diluted to ten parts total: one part sample plus nine parts diluent. Factors multiply along the series. For an awkward target such as 1:2500, factors of 50 and 50 give the exact target without a very small direct aliquot. The first factor is adjustable, and the calculator derives the second.
Set the number of assay replicates, the volume dispensed into each well, and your plate’s working volume limit. The map assigns each dilution across columns or down rows. Replicates share a concentration and are supplied from one preparation tube. Optional diluent blanks occupy the final column or final row; unused wells remain empty. The schedule lists well addresses and concentrations so that the layout can be checked before dispensing.
The volume calculation starts at the last tube and works backward. Each tube must supply its assay wells and the sample needed by the next tube while retaining the entered dead volume and dispensing reserve. Sample transfers round upward to your chosen pipette increment. Diluent quantities follow from those transfers, keeping the requested factors exact. Check that you can dispense those diluent quantities accurately. The recipe reports the mixed volume, outgoing transfer, and residual volume, and flags preparations that exceed the tube limit. Agar inocula are included in the budget when you select a planned tube. A separate reservoir allowance covers the blank wells.
Press play to follow liquid from a source tube into a tube already containing diluent. Mix each prepared dilution before withdrawing the next aliquot. The animation explains the sequence; its timing and liquid heights are schematic.
For colony counting, enter exact counts from one to three agar plates at the same dilution and inoculum volume. Use a planned tube dilution or enter a total denominator independently. The tool divides pooled colonies by the equivalent volume of original sample. It also shows a one-colony equivalent, which helps interpret sparse counts. The FDA BAM Chapter 3 January 2026 range of 15–300 colonies per plate supplies the countability guidance.
This planner assumes additive volumes, homogeneous mixtures, and no analyte loss or growth during preparation. It does not estimate pipetting uncertainty, evaporation, adsorption, assay reagent dilution, or biological recovery. It does not implement the FDA likelihood method for TNTC plates, confidence intervals, or all regulatory reporting rules. A zero count does not establish sterility. Follow the validated method and equipment limits for your experiment.
How it is calculated
Dilution convention and concentration
A denominator D means 1 part sample in D parts total mixture, so Vdiluent = (D − 1) Vsample. It never means 1 part sample plus D parts diluent. ATCC's Introduction to Microbiology gives the 1 mL + 9 mL = 1:10 example. The conservation relation is . Cumulative denominators multiply, including any entered starting dilution; each concentration is the original mass concentration divided by that denominator. The two-step route uses F₂ = target/F₁.
Prepare backward, execute forward
One mixed tube supplies all assay replicates at its dilution. Its required withdrawal Q includes replicate well volume and, when a planned tube is selected for CFU, all agar inocula from that tube. Starting at the final tube with no outgoing serial transfer, require . Choose the smallest sample aliquot on the entered increment grid at or above both this requirement divided by Fⱼ and the pipette minimum. Then Vmix = Fⱼ Vsample and Vdiluent = (Fⱼ − 1) Vsample. Rounding sample upward preserves the exact factor because the diluent follows it.
Prefill each tube with its listed diluent. Add the listed sample from the stock or preceding mixed tube; mix before transferring onward. Finish the entire serial chain before dispensing assay wells and any planned agar inocula. The residual table includes dead volume, dispensing reserve and rounding surplus. The stock load includes one dead-volume allowance. Diluent totals include a shared blank reservoir with replicate demand, excess and one dead-volume allowance. Diluent supply reservoir losses beyond these allowances are not included.
Colony count
FDA BAM Chapter 3, January 2026, pp. 5–7, defines original volume received as inoculum volume divided by the total dilution denominator. For one to three exact counts at the same dilution and inoculum volume, CFU/mL = sum(counts) / sum(original volumes). This is the no-TNTC, equal-volume special case of the FDA estimator. The one-colony equivalent is 1/sum(original volumes), not a confidence limit. Countability guidance uses the chapter's 15–300 per-plate range (p. 3); estimates are displayed without implementing its formal two-significant-digit reporting rule.
Assumptions
Volumes add, mixing is complete before every withdrawal, and analyte concentration changes only by dilution. No growth, adsorption, precipitation, evaporation or transfer loss is modeled. The entered dead volume is a known equipment allowance, not an inferred universal value. Excess is a percentage of all planned dispensing demand at that tube; downstream serial transfers are added exactly. Transfer increments apply to sample aliquots only. Diluent quantities must be accurately dispensable separately. All replicates share one preparation tube. Well limits and tube limits are user-entered working capacities. The assay has no extra reagent volume or subsequent dilution. CFU counts are exact, independent agar plate counts with one common dilution and inoculum volume; they are separate from the assay plate.
Where it applies
This is a preparation planner and arithmetic check, not a validated biological assay or a regulatory reporting system. Confirm pipette calibration, dispenser resolution, adequate mixing and container working limits. Starting-stock pre-dilution preparation is outside the budget. Custom down-row plans have at most 8 stages, or 7 with blanks; across-column plans have at most 12, or 11 with blanks. Counts outside 15–300 are flagged as outside-range estimates under FDA BAM Chapter 3 January 2026. Zero counts do not establish sterility. TNTC plates, multiple dilution levels in one pooled estimate, contaminated plates, spreaders, detection-limit reporting and confidence intervals require the full validated method. No claim of FDA method compliance is made. The animation shows the transfer sequence with schematic liquid levels and time.
References
- FDA, Bacteriological Analytical Manual, Chapter 3: Aerobic Plate Count, January 2026 edition, pp. 3–8. https://www.fda.gov/media/191248/download ; edition index: https://www.fda.gov/food/laboratory-methods-food/bam-chapter-3-aerobic-plate-count
- ATCC, Introduction to Microbiology, Bacterial Enumeration: dilution convention and triplicate CFU example. https://www.atcc.org/resources/culture-guides/introduction-to-microbiology (accessed 26 October 2026). ATCC's 25–250 guidance differs from the newer FDA range used here.
Inputs
| Input | Symbol | Unit | Default |
|---|---|---|---|
| Dilution route A factor of 10 means one part sample in ten parts final mixture, not one plus ten. | route | Repeat one factor | |
| Number of dilution steps Every step has its own preparation tube and assay wells. | n_steps | 6 | |
| Factor per step For a twofold series enter 2; for a tenfold series enter 10. | F_step | 10 | |
| Target denominator (1:D) Total dilution relative to the starting stock; two factors multiply to this target. | D_target | 2500 | |
| First factor in two-step route The second factor is target ÷ first factor. For 1:2500, 50 then 50 is a useful starting point. | F_first | 50 | |
| Starting stock dilution denominator Use 1 for undiluted stock. Include any earlier dilution here; its preparation is outside this volume budget. | D_initial | 1 | |
| Original mass concentration Before the starting dilution. The concentration table uses this mass basis; CFU is calculated separately. | C_original | mg/mL | 1 |
| Step 1 factor Dilution denominator for this step only. | factor_1 | 10 | |
| Step 2 factor Dilution denominator for this step only. | factor_2 | 10 | |
| Step 3 factor Dilution denominator for this step only. | factor_3 | 10 | |
| Step 4 factor Dilution denominator for this step only. | factor_4 | 10 | |
| Step 5 factor Dilution denominator for this step only. | factor_5 | 10 | |
| Step 6 factor Dilution denominator for this step only. | factor_6 | 10 | |
| Step 7 factor Dilution denominator for this step only. | factor_7 | 10 | |
| Step 8 factor Dilution denominator for this step only. | factor_8 | 10 | |
| Step 9 factor Dilution denominator for this step only. | factor_9 | 10 | |
| Step 10 factor Dilution denominator for this step only. | factor_10 | 10 | |
| Step 11 factor Dilution denominator for this step only. | factor_11 | 10 | |
| Step 12 factor Dilution denominator for this step only. | factor_12 | 10 | |
| Dilution direction Replicates occupy rows A onward when across, and columns 1 onward when down. | orientation | Across columns (1 → 12) | |
| Assay replicates per dilution One preparation tube supplies all replicates at that dilution. | replicates | 3 | |
| Dispense per assay well No later assay reagent addition is included. This is also the final well volume. | V_well | uL | 100 |
| Well working volume limit Set from your plate datasheet. This is a working limit, not a universal 96-well capacity. | V_well_max | uL | 300 |
| Reserve a diluent blank per replicate Blanks use column 12 when across or row H when down. Other unassigned wells remain empty. | blank_wells | true | |
| Dispensing excess (%) Reserve this percentage of the assay dispense demand in every tube and in the shared blank reservoir. | excess_pct | 10 | |
| Dead volume per source container Unrecoverable liquid left in each preparation tube, starting stock container and shared blank reservoir. | V_dead | uL | 50 |
| Minimum reliable sample transfer Use the minimum reliable volume of your pipette. Larger factors may require larger preparation tubes. | V_min | uL | 2 |
| Sample transfer increment Sample aliquots round up to this increment. Diluent volumes remain exact; use a suitable dispenser. | V_increment | uL | 1 |
| Preparation tube working limit Allows headspace for mixing; applies to mixed preparation volumes, not stock or diluent reservoirs. | V_tube_max | uL | 1500 |
| Colony plate dilution A separate agar plate, not the assay wells. Selecting a planned tube includes all agar inocula in its preparation volume budget. | cfu_basis | Enter total dilution independently | |
| Agar plate sample dilution (1:D) Include every dilution before agar inoculation. Plated volume is entered separately. | D_cfu_manual | 100000 | |
| Tube used to inoculate agar Includes the starting stock dilution denominator. | count_stage | 2 | |
| Inoculum per agar plate Sample volume put on each agar plate, excluding agar medium. | V_plated | mL | 0.1 |
| Agar plates at this dilution Counts are pooled only across the same dilution and inoculum volume. | count_plates | 1 | |
| Colonies on agar plate 1 Enter an exact count. TNTC and contaminated plates are outside this calculator. | colonies_a | 150 | |
| Colonies on agar plate 2 Enter an exact count. | colonies_b | 140 | |
| Colonies on agar plate 3 Enter an exact count. | colonies_c | 160 | |
| Transfer animation ▶ Each interval shows one transfer into a prefilled tube. Animation is schematic, not elapsed laboratory time. | transfer_phase | 2.35 |
Results
With the default inputs above, the calc gives:
| Result | Value |
|---|---|
| Starting stock to load (includes dead volume) | 93 uL |
| Total diluent to prepare | 2,648 uL |
| Largest mixed preparation tube | 430 uL |
| Final dilution denominator (1:D) | 1,000,000 |
| Assigned wells, including blanks | 21 |
| Original sample (CFU/mL) | 150,000,000 1/mL |
| One-colony equivalent (CFU/mL) | 1,000,000 1/mL |
Checked against sources
Each case below was calculated by this calc and agrees with the value from its source (within the tolerance where one is shown).
FDA BAM: 150 colonies, 1:100, 1 mL → 15,000 CFU/mL
Source: FDA BAM Chapter 3, January 2026, p. 5: 150 CFU in 0.01 mL original homogenate gives 15,000 CFU/mL. https://www.fda.gov/media/191248/download
Inputs: Agar plate sample dilution (1:D) = 100; Inoculum per agar plate = 1 mL; the others as above.
| Result | Source | This calc |
|---|---|---|
| Original sample volume per agar plate | 0.01 mL | 0.01 mL |
| Original sample (CFU/mL) | 15,000 1/mL | 15,000 1/mL |
FDA BAM Example 2.2a: 27 colonies → 2,700 CFU/mL
Source: FDA BAM Chapter 3, January 2026, Example 2.2a, p. 7: 27 CFU, 1:100 dilution, 1 mL inoculum. https://www.fda.gov/media/191248/download
Inputs: Agar plate sample dilution (1:D) = 100; Inoculum per agar plate = 1 mL; Colonies on agar plate 1 = 27; the others as above.
| Result | Source | This calc |
|---|---|---|
| Original sample (CFU/mL) | 2,700 1/mL | 2,700 1/mL |
FDA BAM Example 2.1b: equal-volume duplicate plates
Source: FDA BAM Chapter 3, January 2026, Example 2.1b, p. 6. Equal original volumes of 0.01 mL and per-plate concentrations 2700 and 5100 CFU/mL correspond to counts 27 and 51; pooled result 3900 CFU/mL. https://www.fda.gov/media/191248/download
Inputs: Agar plate sample dilution (1:D) = 100; Inoculum per agar plate = 1 mL; Agar plates at this dilution = 2; Colonies on agar plate 1 = 27; Colonies on agar plate 2 = 51; the others as above.
| Result | Source | This calc |
|---|---|---|
| Total original sample volume counted | 0.02 mL | 0.02 mL |
| Original sample (CFU/mL) | 3,900 1/mL | 3,900 1/mL |
ATCC triplicate example: 40, 37, 43 → 4.0 × 10⁸ CFU/mL
Source: ATCC Introduction to Microbiology, Bacterial Enumeration. Counts 40, 37, 43 at a final plated dilution of 10⁻⁷ yield 4.0 × 10⁸ CFU/mL. Here sample dilution is 10⁻⁶ and the separately entered 0.1 mL plated volume supplies the additional tenth; entering 10⁻⁷ as the sample dilution would double-count that volume. https://www.atcc.org/resources/culture-guides/introduction-to-microbiology
Inputs: Agar plate sample dilution (1:D) = 1,000,000; Agar plates at this dilution = 3; Colonies on agar plate 1 = 40; Colonies on agar plate 2 = 37; Colonies on agar plate 3 = 43; the others as above.
| Result | Source | This calc |
|---|---|---|
| Mean colonies per agar plate | 40 | 40 |
| Original sample (CFU/mL) | 400,000,000 1/mL | 400,000,000 1/mL |
ATCC dilution convention: 1 part sample + 9 parts diluent
Source: ATCC Introduction to Microbiology: 1.0 mL sample mixed with 9.0 mL diluent is a one-to-ten dilution. This check verifies the same ratio at the planner's automatically sized preparation volume. https://www.atcc.org/resources/culture-guides/introduction-to-microbiology
Inputs: Number of dilution steps = 1; the others as above.
| Result | Source | This calc |
|---|---|---|
| Final dilution denominator (1:D) | 10 | 10 |
| Diluent-to-sample ratio in the first tube | 9 | 9 |
Responses