CFU per mL Calculators

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CFU per mL (colony-forming units per milliliter) measures the concentration of viable, culturable bacteria or fungi in a liquid sample. Each colony on an agar plate represents one viable cell (or clump). CFU/mL = number of colonies / (dilution factor × volume plated in mL). For reliable results, plates should contain 30–300 colonies. Serial dilution of the sample brings colony counts into this reliable range. Unlike OD₆₀₀, which measures total (live + dead) cells, CFU counts only cells capable of dividing to form a visible colony.

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CFU/mL Formula

CFU/mL = colonies / (dilution factor × volume plated mL)

Example: 68 colonies on 10⁻⁵ dilution plate, 0.1 mL plated: CFU/mL = 68 / (10⁻⁵ × 0.1) = 68 / 10⁻⁶ = 6.8 × 10⁷ CFU/mL.

Serial Dilution

Transfer 1 mL into 9 mL diluent (10⁻¹); repeat to 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵. Mix each tube thoroughly (vortex 5 s) before transferring. Plate 0.1 mL from multiple dilutions.

30–300 Colony Rule

  • 30–300: statistically reliable range; use for calculation
  • < 30 (TFTC): too few to count — unreliable
  • > 300 (TNTC): too numerous — colonies merge, undercount

Good Practice

Plate duplicates from each dilution; average counts if SD < 15%. If > 15%: plating error; repeat. Report mean ± SD with dilution used.

Glossary

CFU/mL
Colony-forming units per milliliter; viable culturable cell count: CFU/mL = colonies / (dilution × volume); uses 30–300 colony plates; counts only cells capable of forming visible colonies.
30-300 Colony Rule
Only plates with 30–300 colonies are used for CFU/mL calculation; < 30 = TFTC (statistically unreliable); > 300 = TNTC (colonies overlap and undercount).
Serial Dilution
Sequential 1:10 dilution steps (1 mL into 9 mL diluent); reduces sample to countable range; total dilution = 10^(-n); vortex each tube thoroughly before transferring.

Frequently Asked Questions

CFU/mL = colonies / (dilution factor × volume plated). Step 1: Serial dilutions of sample (10⁻³ to 10⁻⁷). Step 2: Plate 0.1 mL from multiple dilutions onto agar. Step 3: Incubate at appropriate temperature and time. Step 4: Count colonies on plates with 30–300 colonies. Step 5: Apply formula. Example: 145 colonies on 10⁻⁶ dilution, 0.1 mL plated: CFU/mL = 145 / (10⁻⁶ × 0.1) = 1.45 × 10⁹ CFU/mL. Always plate duplicates and report the mean ± SD.

Below 30 (TFTC): each missed or miscounted colony represents > 3% relative error — statistically unreliable. Above 300 (TNTC): colonies physically overlap and merge, preventing accurate counting; nutrient depletion also produces abnormally small colonies. The 30–300 range: provides Poisson statistical reliability (< 10% relative error) while keeping colonies well-separated and visually distinct. If no plate falls in range: report as estimated (< lower limit or > upper limit) and redesign the dilution scheme.

Serial dilution is step-wise 1:10 dilution of a sample to reduce bacterial concentration to a countable range. Necessary because environmental and clinical samples often have 10⁵–10⁹ CFU/mL — too dense to plate directly. Steps: 1 mL sample into 9 mL diluent (1:10 = 10⁻¹); vortex; 1 mL from this tube into 9 mL (10⁻²); repeat to needed depth. Critical: vortex each tube before transferring — poor mixing is the most common cause of inaccurate CFU counts.

CFU/mL counts only viable, culturable cells — those that grow and divide to form visible colonies. Excludes dead cells, VBNC (viable but non-culturable) cells, and counts clumps as 1 CFU regardless of how many cells they contain. Total cell count uses microscopy with fluorescent stains (DAPI, acridine orange) or flow cytometry to count all cells (live + dead + VBNC). Total count is typically 2–100× higher than CFU count. Use CFU when measuring antibiotic kill, food safety testing, or quantifying viable organisms. Use total count for total biomass or when culturing conditions underrepresent the full population.