Counting bacteria you cannot see
If you work in a microbiology lab, one question comes up constantly: how many living bacteria are actually in this sample? You cannot count them one by one under a microscope and get a meaningful number, because a milliliter of a bacterial culture can hold hundreds of millions of cells. The practical answer is the colony-forming unit, or CFU. You spread a tiny, known amount of a diluted sample on an agar plate, let it incubate, and count the colonies that grow. Each colony started from one living cell or a small clump of cells, so the colony count tells you the concentration of viable bacteria in the original sample. This calculator turns that plate count into a standard CFU per milliliter value.
The formula behind the count
Formula: CFU per mL = colonies counted x dilution factor / volume plated (mL)
Each part is straightforward. The colonies counted is the number of colonies on your plate. The dilution factor is how much you diluted the original sample before plating, written as a whole number: a 1 to 1000 dilution has a factor of 1000. The volume plated is the amount of diluted sample you spread on the plate, in milliliters, usually 0.1 mL. Multiply colonies by the dilution factor and divide by the plated volume, and the result is the concentration in the original, undiluted sample.
How to use this calculator
- Count the colonies on the plate and enter that number.
- Enter the dilution factor of the sample you plated.
- Enter the volume you spread on the plate, in milliliters.
- Read the CFU per mL result.
Worked example
Suppose you count 60 colonies on a plate. The sample was diluted 1 to 1000, so the dilution factor is 1000, and you plated 0.1 mL of the diluted sample. Step 1: multiply colonies by the factor, 60 x 1000 = 60,000. Step 2: divide by the plated volume, 60,000 / 0.1 = 600,000. The original sample contained 600,000 colony-forming units per milliliter, or 6.0 x 10^5 CFU/mL. If you want to sanity-check the chemistry side of your lab work, the grams to moles calculator and the mole fraction calculator handle solution-side math, and the mL to moles calculator covers the concentration conversions behind your dilutions.
Where CFU counts matter
Food safety labs use CFU counts to check whether milk, meat, or packaged foods stay within legal bacterial limits. Water treatment plants test drinking water and swimming pools the same way. Pharmaceutical companies validate that sterile products are genuinely sterile, and hospitals track contamination on surfaces and in air samples. Anywhere a living microbe count decides a pass or fail result, this calculation is the standard.
Mistakes that ruin a plate count
- Counting crowded plates. Plates with more than about 300 colonies merge together and undercount; plates with fewer than 30 colonies are statistically shaky. Aim for the 30 to 300 range by choosing the right dilution.
- Using the wrong dilution factor. A 1 to 100 dilution is a factor of 100, not 0.01. Mixing this up throws the answer off by orders of magnitude.
- Counting too early. Slow growers need the full incubation time. Count before that and colonies are invisible or too small to see.
An honest limitation
A CFU count only reflects cells that are alive and able to grow on your chosen medium under your chosen conditions. Dead cells, viable-but-non-culturable cells, and microbes that need different conditions never appear. Treat the result as an estimate of culturable bacteria, which is what food, water, and pharma standards are written around.