Colony Counting Calculators

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Colony counting is the process of enumerating bacterial or fungal colonies on agar plates to determine the concentration of viable organisms in a sample (CFU/mL or CFU/g). Each colony is assumed to have originated from one viable cell or cell clump (colony-forming unit, CFU). CFU/mL = colonies counted / (dilution factor × volume plated in mL). Accurate colony counting requires: selecting plates with 30–300 colonies (for bacteria) or 15–150 (for fungi); proper serial dilution to bring counts into this range; consistent colony identification; and duplicate plating. Colony counting is used in food safety, clinical microbiology, pharmaceutical manufacturing, and research.

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CFU/mL Formula

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

Example: 127 colonies on a 10⁻⁵ dilution plate, 0.1 mL plated: CFU/mL = 127 / (10⁻⁵ × 0.1) = 127 / 10⁻⁶ = 1.27 × 10⁸ CFU/mL.

Acceptable Colony Count Range

  • Bacteria: 30–300 per plate (statistically reliable, colonies distinct)
  • Fungi/mold: 15–150 per plate (colonies are larger)
  • < 30 = TFTC (too few to count); report as estimated
  • > 300 = TNTC (too numerous to count); report as estimated

Counting Methods

Manual: mark counted colonies with a marker; use a colony counter (magnifying lens with dark background and grid). Automated: colony counting software (ImageJ, OpenCFU, Scan500); camera captures plate image; software counts circles or blobs. Accuracy: count all distinct colonies including tiny ones; don't count confluent growth.

Key Rules

Count duplicate plates from each dilution → average. If SD between duplicates > 15%: plating error; repeat. Report results as mean ± SD with dilution noted.

Glossary

Colony-Forming Unit (CFU)
A single colony on an agar plate assumed to originate from one viable cell or cell cluster; CFU/mL = colonies/(dilution × volume); counts only culturable viable cells.
30-300 Rule
Only plates with 30–300 colonies (15–150 for fungi) are used for CFU/mL calculation; < 30 = TFTC (statistically unreliable); > 300 = TNTC (colonies overlap and undercount).
Serial Dilution
Sequential 1:10 dilutions (1 mL into 9 mL diluent) reducing sample concentration to the countable 30–300 colony range; vortex each tube before transferring; total dilution = 10^(−n steps).

Frequently Asked Questions

CFU/mL = colonies / (dilution factor × volume plated mL). Step 1: perform serial dilutions of the sample (10-fold steps). Step 2: plate 0.1 mL from each dilution on agar plates in duplicate. Step 3: incubate at appropriate temperature and time. Step 4: count colonies on plates with 30–300 colonies (bacteria) or 15–150 (fungi). Step 5: calculate CFU/mL. Example: 85 colonies on 10⁻⁶ dilution plate, 0.1 mL: CFU/mL = 85 / (10⁻⁶ × 0.1) = 8.5 × 10⁸ CFU/mL. Average duplicate plates from the same dilution. If multiple dilutions are in the 30–300 range, average the CFU/mL estimates from each.

Plates with 30–300 colonies give the most statistically reliable CFU/mL estimates: Below 30 colonies (TFTC): each missed or miscounted colony has a large relative effect on the result. With 10 colonies, one miscount = 10% error; with 30 colonies, one miscount ≈ 3.3% error. Above 300 colonies (TNTC): colonies overlap and merge → undercounting; nutrient depletion may cause abnormally small colonies; spatial competition among colonies. 30–300 range: sufficient numbers for Poisson counting statistics to give < 10% relative error; colonies are separated and individually countable. For fungi: 15–150 because fungal colonies are larger and may be less distinct at higher densities.

TNTC (too numerous to count, > 300 colonies): report as 'TNTC'; provide estimate: if all dilutions are TNTC, use the highest dilution plate and estimate CFU/mL as > (300 / dilution × volume). Redesign dilution scheme for next experiment with higher dilutions. TFTC (too few to count, < 30 colonies): report as 'TFTC'; provide estimate: if all dilutions are TFTC, use the lowest dilution plate and estimate as < (30 / dilution × volume). Redesign with lower dilutions. No dilution in range: redesign the serial dilution series — if you have no plates in the 30–300 range, results cannot be reliably quantified. Document: always report which plates were counted, which were TNTC/TFTC, and the dilution used.

Spread plate method: sample is placed on the surface of already solidified agar; spread evenly using a sterilized L-shaped spreader or glass beads; colonies grow on the surface — easy to pick; preferred for organism isolation. Pour plate method: sample is mixed with molten agar (~45°C) in a tube; poured into petri dish; agar solidifies with cells embedded; colonies grow inside and on the surface — some colonies may be subsurface (small, lenticular) and harder to count; useful for anaerobe counting under reduced O₂ conditions. Colony morphology difference: surface colonies appear circular with defined edges; subsurface colonies appear as small lenticular (lens-shaped) dots. For food microbiology: spread plating is generally preferred for aerobic plate count; pour plating is used when low melting point agar is required (some selective media).