How to Count Bacterial Colonies on Agar Plates

A colony is a visible cluster of microorganisms growing on solid media, and in the standard plate count we assume each colony grew from a single viable cell (or a single colony forming unit). Counting colonies accurately is therefore how microbiologists estimate how many living cells were in the original sample. The method below is the one used in food, water, clinical and research labs worldwide.

What you need before counting

• An incubated agar plate with visible, well-separated colonies.
• The dilution factor and the volume plated (for the CFU calculation).
• A counting aid — a marker and tally counter, a colony counting grid, or an online colony counter that marks colonies from a photo.

Step 1: Pick a plate in the countable range

Not every plate gives a reliable number. Plates with too few colonies are dominated by random variation, while overcrowded plates have colonies that merge and hide one another. The accepted compromise is the 30–300 rule: count plates with between 30 and 300 colonies. If you ran a dilution series, choose the dilution whose plate falls in this window.

Step 2: Count systematically so nothing is missed or doubled

Place the plate on a dark or contrasting background and good light. Work across it in a fixed pattern — for example, dividing the dish into quadrants or following a grid — and mark each colony as you count it so you never tally the same one twice. A bench colony counter with a magnifier and a felt-tip pen on the base of the dish is the classic manual approach; counting from a photo with software achieves the same thing with less eye strain.

Handling difficult colonies

• Touching colonies: count them separately when each has a distinct centre and edge; count them as one when they have fully merged.
• Spreaders: a colony that spreads in a thin film is counted as a single colony, not as the many shapes it forms.
• Edge colonies: apply one rule consistently, such as counting colonies that touch the edge on only two adjacent sides.
• Pinpoint colonies: use magnification and good lighting so very small colonies are not overlooked.

Step 3: Record the colony count with its dilution

A colony count is only meaningful alongside the dilution it came from. Note the number of colonies, the dilution factor of that plate, and the volume you plated. These three numbers are everything you need for the next step.

Step 4: Convert the count into CFU/mL

The colony count is reported as colony forming units (CFU). To get the concentration in the original sample, divide the colony count by the product of the dilution factor and the plated volume. The full worked formula, with examples, is in our guide on how to calculate CFU per mL.

Manual counting vs. an online colony counter

Manual counting works, but it is slow on dense plates and varies from person to person. Counting from a photograph with an automatic colony counter is faster, applies the same logic to every plate, and gives you a permanent image record of what was counted. For routine plate counts, that consistency is often worth more than the raw speed. You can try this approach with the free online colony counter on the homepage.

Common mistakes to avoid

• Counting plates outside the 30–300 range and reporting the number anyway.
• Changing your edge or merged-colony rule from plate to plate.
• Forgetting to record the dilution factor with the count.
• Poor lighting that hides pinpoint colonies or creates glare.