# Count bacterial colonies in ImageJ (or from a plate photo, free online)

> Count colonies in ImageJ: oval on the dish, threshold, Watershed, Analyze Particles. Then CFU/mL from the count and dilution, with a worked example.

Published 2026-11-06 by LabCanvas team. https://www.labcanvas.com/blog/colony-counter-online

![ImageJ counting colonies on a synthetic photo of a 60 mm dish: Analyze Particles outlines and numbers 60 colonies, and the Summary table gives a count of 60 and an average size of 1.275 mm²](https://www.labcanvas.com/blog/imagej/colony-counter-online-result.webp)

To count colonies in ImageJ, set the scale from the dish, draw an oval just inside the rim and clear everything outside it, threshold the colonies (**Image › Adjust › Threshold…**), split touching ones with **Process › Binary › Watershed**, and run **Analyze › Analyze Particles…** with a minimum size and **Summarize** ticked. Then CFU/mL = colonies ÷ (volume plated in mL × dilution): 60 colonies from 100 µL of a 10⁻⁵ dilution is 6.0 × 10⁷ CFU/mL.

## The plate in this example

A synthetic photo of a 60 mm dish, 720 × 720 pixels, made for testing: 60 dark colonies on light agar, 0.3 to 3.4 mm² each, including five pairs that touch, with the uneven lighting and the darker edge you get from a real phone photo. Knowing the true count lets you check every step.

## How do I count colonies in ImageJ? Step by step

These commands are in ImageJ 1.x, so they are the same in Fiji.

1. **Open the photo** (File › Open). JPEG from a phone is fine for counting; keep the original.
2. **Set the scale from the dish.** Draw a straight line across the dish from inner wall to inner wall through the centre, then **Analyze › Set Scale…**: Known distance **60** (or your dish's inner diameter), Unit **mm**. Here the line was 636 pixels, so 0.0943 mm per pixel. Zoom in before you click the ends; one pixel off at each end is 0.2 mm on the diameter. (Details: [how to set the scale in ImageJ](https://www.labcanvas.com/blog/imagej-scale-bar-set-scale).)
3. **Image › Type › 8-bit.** For coloured colonies on coloured agar, **Image › Color › Split Channels** and keep the channel with the most contrast is often better (here the green channel: agar about 214, colonies about 52).
4. **Keep only the inside of the dish.** Pick the oval tool, Shift-drag a circle a little inside the rim (here 600 pixels across on a 636-pixel dish, about 1.7 mm in from the wall), then **Edit › Clear Outside**. The rim, its shadow and the bench are gone; with white as the background colour (**Edit › Options › Colors…**) they no longer look like colonies.
5. **Threshold.** **Image › Adjust › Threshold…**, method **Default**, **Dark background** off (dark colonies on light agar; tick it for light colonies on dark agar). Check that every colony is red and the agar is not. Here Default chose 0–153, 3.06% of the image. Click **Apply**.
6. **Clean up the mask.** **Process › Binary › Convert to Mask** (if Apply did not already), **Process › Binary › Fill Holes** (colonies with a pale centre), then **Process › Binary › Watershed** to split touching colonies.
7. **Count.** **Analyze › Analyze Particles…**, Size **0.1–Infinity** mm² (drops specks of dust and agar texture), Circularity 0.00–1.00, Show **Outlines** or **Overlay**, **Summarize** ticked, and **Add to Manager** if you want to check the outlines on the colour photo.

![ImageJ Threshold window over a cleared dish with the colonies in red](https://www.labcanvas.com/blog/imagej/colony-counter-online-threshold.webp "ImageJ 1.54f on the synthetic plate, after 8-bit, an oval inside the rim 1 and Clear Outside. Image › Adjust › Threshold: Default method 2, Dark background off 3, so 0–153 is selected 4 and the colonies are red, 3.06% of the image 5.")

![ImageJ Summary table counting 60 colonies, with numbered outlines on the plate](https://www.labcanvas.com/blog/imagej/colony-counter-online-result.webp "After Convert to Mask, Fill Holes, Watershed and Analyze Particles from 0.1 mm²: the Summary counts 60 colonies the true number, 76.522 mm² in total, 1.275 mm² on average. The outlines, from the ROI Manager, are drawn on the original photo to check them.")

On this plate, ImageJ found 60 colonies, all of them. Without Watershed it found 55: each of the five touching pairs counted as one.

![Five pairs of touching colonies before and after ImageJ Watershed](https://www.labcanvas.com/blog/imagej/colony-counter-online-watershed.webp "The five touching pairs on the sample plate, outlined by Analyze Particles. Top: without Watershed each pair is one particle 55 colonies in all. Bottom: after Process › Binary › Watershed each pair is split into two 60.")

### Why does Analyze Particles count the rim, or nothing at all?

- **The rim or the bench is counted:** you skipped Clear Outside, or the oval touches the wall. Draw it a little smaller.
- **One huge particle, or a count of 0:** the threshold selected the agar, not the colonies. Flip **Dark background**, or invert the mask with **Edit › Invert**.
- **Hundreds of tiny particles:** the minimum size is too small, or the threshold reaches into the agar's texture. Raise the size to the smallest real colony (about a fifth of a typical one) or blur slightly first (**Process › Filters › Gaussian Blur…**, sigma 1).
- **Colonies near the edge are missed:** the light falls off towards the wall. Subtract the background first (**Process › Subtract Background…**, light background ticked, radius larger than the biggest colony), or threshold each half separately.

## How do I calculate CFU/mL from the count?

A colony-forming unit (CFU) is one viable cell, or clump of cells, that grew into a colony. The formula:

$$\text{CFU/mL} = \frac{\text{number of colonies}}{\text{volume plated in mL} \times \text{dilution factor of that tube}}$$

Worked example with this plate: you made a tenfold serial dilution of an overnight culture, spread **100 µL** of the **10⁻⁵** tube, and counted **60** colonies.

| Step | Value |
|---|---|
| Colonies counted | 60 |
| Volume plated | 100 µL = 0.1 mL |
| Dilution of the plated tube | 10⁻⁵ |
| CFU/mL in the original culture | \(60 \div (0.1 \times 10^{-5}) = \mathbf{6.0 \times 10^7}\ \textbf{CFU/mL}\) |

Plan the series with the [dilution calculator](https://www.labcanvas.com/tools/dilution-calculator) (its serial dilution planner gives the volumes tube by tube).

Some rules that keep the number honest:

- **Count plates in the countable range**, about 30 to 300 colonies on a standard 90–100 mm plate (some standards use 25–250). Below it, chance dominates; above it, colonies merge and the count falls short. On a 60 mm dish, the top of the range is lower.
- **Average the replicate plates of the same dilution** before you apply the formula, and if two consecutive dilutions are both in range, check that they agree within about tenfold.
- **Report it as CFU/mL (or CFU/g), never as "cells"**: a chain or a clump gives one colony.
- With too few colonies, say so: "fewer than 30 colonies; estimated count" rather than a precise figure.

## How should I photograph the plates?

The count is only as good as the photo.

- **Lid off**, or the lid's reflections and condensation look like colonies.
- **Straight above**, camera parallel to the plate. A tilted phone makes the dish an ellipse and the scale wrong across it. A copy stand or a box with a hole in the lid helps.
- **Even light**: a light box underneath for clear agar, or diffuse light from the side on a dark background for opaque agar. Avoid a ring light's reflection in the middle of the agar.
- **The whole dish, filling the frame**, and the same zoom, light and exposure for every plate in the experiment.
- **A scale**: the dish's inner diameter is enough; a ruler beside the dish is better.
- Label each plate in the picture (on the side of the base), so photo and plate cannot be mixed up.

## When is a count by hand better than ImageJ?

Swarming or spreading colonies, colonies of several morphologies to score separately, or plates with hundreds of tiny colonies defeat a threshold. Count those by hand: in Fiji, **Plugins › Analyze › Cell Counter** keeps a tally per marker type as you click; in plain ImageJ, the **Multi-point** tool numbers each click and **Analyze › Measure** gives the count. OpenCFU (Geissmann 2013) is a free desktop colony counter built for this job, and a Fiji macro that repeats the steps above is the route when you have thousands of plates and want the whole pipeline scripted.

## Pitfalls

- **Merged colonies.** Watershed splits round pairs; it cannot split a spreading lawn. If more than a few colonies touch, plate a higher dilution.
- **Debris and bubbles.** Air bubbles in the agar and dust look like small dark colonies. The minimum size drops most of them; look at the outlines on the photo before you trust the count.
- **Colonies on the wall.** Colonies growing up the side of the dish are hidden in the rim. Count those by eye and add them, or accept a small, consistent undercount and say so.
- **Different settings per plate.** Choose the threshold method and size limit on a few plates, then keep them for the whole experiment.
- **Never checked.** Count three plates by hand and compare with the automatic count before you rely on it.

## Reporting it

> Plates were photographed from above on a [light box] with a [phone camera] at a fixed height. Colonies were counted in [Fiji (ImageJ 1.54)]: scale from the [60] mm dish, 8-bit, an oval region inside the rim, [Default] threshold, Fill Holes, Watershed and Analyze Particles from [0.1] mm²; automatic counts were checked against manual counts on [3] plates. Plates with 30–300 colonies were used, and CFU/mL was calculated as colonies ÷ (volume plated × dilution).

Cite ImageJ (Schneider et al. 2012) or Fiji (Schindelin et al. 2012). Plot CFU/mL on a log axis, with each biological replicate as a point.

## An easier way: count colonies in LabCanvas

The same count runs in the browser in [LabCanvas Image Analysis](https://www.labcanvas.com/analysis.html), with nothing to install and no oval to draw: its Colonies assay finds the dish for you.

1. Open the plate photo, or click **Try it on a sample** (this plate is built in).
2. In the **Assays** tab, choose **Colony counting**.
3. Keep **Round dish** (it finds the dish and leaves out the rim), pick dark-on-light colonies, set the **Smallest colony** in mm², and leave **Fill holes** and **Split touching colonies** on.
4. Read the count, colonies per cm², the mean size and the area covered; tick **List every colony** for each colony's size.

![Colony counting in LabCanvas Image Analysis](https://www.labcanvas.com/analysis-examples/colonies.webp "The same synthetic plate in the LabCanvas Colonies assay: the dish found automatically blue circle, outer 4% left out, Otsu threshold on brightness, Fill holes and Split touching colonies. 60 colonies, 2.21 per cm², mean size 1.26 mm², 2.79% of the dish covered.")

On this plate both programs found all 60 colonies. The mean sizes differ a little (1.275 mm² in ImageJ with the Default threshold, 1.26 mm² in LabCanvas with Otsu) because the threshold sets where each colony's edge is. ImageJ's %Area (1.659) is a share of the whole picture; LabCanvas gives a share of the dish (2.79%).

Why it is a good choice for plate counts:

- **Nothing to install**: it runs in the browser on any computer or tablet, so you can count at the bench.
- **No manual steps**: the dish is found, the rim left out and touching colonies split in one assay, instead of an oval, Clear Outside and four binary commands.
- **An image check first**: it flags JPEG compression, clipped pixels and uneven light before you count.
- **Straight into Graphs**: **Run on many images** gives one row per plate and **Send to Graphs** plots the counts per condition. Counting, **Copy** and **Send to Graphs** are free with a LabCanvas account; downloading CSV files is part of LabCanvas Pro.
- **Checked**: every measurement is validated in [more than 4,000 automated tests](https://www.labcanvas.com/image-analysis-validation).

Swarming colonies still need a count by hand; the **Cell counter** in the **Count** tab counts clicks in named categories. [Open Image Analysis](https://www.labcanvas.com/analysis.html) to count your own plates. ImageJ and Fiji are independent open-source projects; LabCanvas is not affiliated with them.

## References

- Geissmann Q (2013). OpenCFU, a new free and open-source software to count cell colonies and other circular objects. *PLoS ONE* 8, e54072. [doi:10.1371/journal.pone.0054072](https://doi.org/10.1371/journal.pone.0054072)
- Schindelin J, Arganda-Carreras I, Frise E, et al. (2012). Fiji: an open-source platform for biological-image analysis. *Nature Methods* 9, 676–682. [doi:10.1038/nmeth.2019](https://doi.org/10.1038/nmeth.2019)
- Schneider CA, Rasband WS, Eliceiri KW (2012). NIH Image to ImageJ: 25 years of image analysis. *Nature Methods* 9, 671–675. [doi:10.1038/nmeth.2089](https://doi.org/10.1038/nmeth.2089)

## Frequently asked questions

### How do I count colonies in ImageJ?

Set the scale from the dish diameter, convert to 8-bit, draw an oval just inside the rim and use Edit › Clear Outside, threshold with Image › Adjust › Threshold (Dark background off for dark colonies on light agar), Convert to Mask, Fill Holes and Watershed, then Analyze › Analyze Particles with a minimum size and Summarize ticked. The Summary window gives the count.

### How do I calculate CFU/mL from a colony count?

CFU/mL = colonies ÷ (volume plated in mL × dilution). For 60 colonies from 100 µL of a 10⁻⁵ dilution: \(60 \div (0.1 \times 0.00001) = 6.0 \times 10^7\ \text{CFU/mL}\). Use plates with roughly 30 to 300 colonies.

### Why does ImageJ count two touching colonies as one?

Touching colonies form one connected blob in the mask. Process › Binary › Watershed cuts round blobs apart along their narrowest point; on our sample plate it raised the count from 55 to the true 60. Very irregular or spreading colonies are better counted by hand.

### Is there a free colony counter online?

Yes. LabCanvas Image Analysis has a Colonies assay that runs in the browser, finds the dish and counts the colonies, free with a LabCanvas account (see the last section). OpenCFU is a free desktop program for the same job.

### How should I photograph plates for counting?

Lid off, camera straight above and parallel to the plate, evenly lit from the side or from below on a dark background, no reflections on the agar, the whole dish in the frame, and the same settings for every plate. Put a ruler beside the dish or use its known diameter to set the scale.

### Can ImageJ count colonies on a plate with hundreds of colonies?

It can, but accuracy drops as colonies merge into each other, and plates above about 300 colonies are outside the range a plate count should use anyway. Plate a higher dilution instead, and check a few automatic counts against a count by hand.

## Related links

- [Dilution calculator](https://www.labcanvas.com/tools/dilution-calculator): Plan the serial dilution tube by tube
- [How to count cells in ImageJ](https://www.labcanvas.com/blog/count-cells-imagej): Threshold, watershed and Analyze Particles
- [Analyze Particles in ImageJ, explained](https://www.labcanvas.com/blog/analyze-particles-imagej): Every option in the dialog
- [Watershed for touching particles](https://www.labcanvas.com/blog/watershed-touching-particles): When it helps and when it cuts
- [Image Analysis: the complete guide](https://www.labcanvas.com/blog/image-analysis-complete-guide): Every tool and setting in the app