What Is a Colony Forming Unit (CFU)?
A colony forming unit (CFU) is a measure of viable bacterial or fungal cells in a sample. Unlike direct cell counts (e.g., hemocytometer or spectrophotometry), CFU counts only living cells capable of dividing and forming a visible colony on agar media.
The term "colony forming unit" rather than "cell" is deliberate — a single colony may originate from one cell or from a clump of cells. CFU counts therefore represent a lower bound on the true number of viable organisms.
CFU enumeration is the gold standard for viability measurement in microbiology, food safety, pharmaceutical QC, and environmental monitoring. FDA BAM, USP, and ISO 4833 all specify plate count methods as primary assays.
How Serial Dilution Works
Serial dilution is a stepwise reduction of sample concentration. Starting from the neat (undiluted) sample, each transfer dilutes the previous solution by a fixed factor — typically 1:10 (tenfold).
For a 1:10 serial dilution: transfer 1 mL of sample into 9 mL of diluent, mix thoroughly, then transfer 1 mL of that tube into the next 9 mL tube. Each step increases the dilution by one order of magnitude: 10−1, 10−2, 10−3, and so on.
Plating multiple dilutions ensures that at least one dilution yields a countable number of colonies. The optimal range balances statistical precision (enough colonies to reduce sampling error) against colony crowding (too many causes merger and undercounting).
Always plate at least 3–4 consecutive dilutions in duplicate. This guarantees at least one dilution falls within the countable range, even if your initial estimate of sample concentration is off by 1–2 orders of magnitude.
Best Practices for Accurate Plate Counts
Vortex or shake each dilution tube for at least 5 seconds before transferring. Incomplete mixing is the most common source of inter-replicate variability and can bias counts by 50% or more.
Use fresh pipette tips for each transfer — carryover from higher concentrations can artificially inflate counts at higher dilutions. Pre-wet tips by pipetting up and down once before the transfer.
Record counts within the recommended incubation window (typically 24–48 hours at 35–37°C for mesophilic bacteria). Extended incubation allows pinpoint colonies to emerge and can inflate counts. Document the exact incubation time and temperature.
When replicates at the same dilution disagree by more than 2-fold, investigate: uneven agar thickness, inadequate mixing, contaminated diluent, or systematic pipetting error. Do not simply average discordant replicates — they indicate a methodological problem.
