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Counting Colonies or Plaques Directly from Images of Agar Plates

background values that may vary across a counting region. The Coarse setting is recommended if colonies or plaques are tightly clustered, providing more accurate detection within these regions. The Extra Fine setting is particularly good to use when there is large variability in the background across the counting region. This setting can be used to reduce or eliminate the detection of false colonies or plaques along plate edges.

The software is equipped with an adjustable counting threshold for each color group. The threshold value corresponds to the pixel group intensity above background that must be achieved in order for a counting zone to be defined as a colony or plaque. As the counting threshold is increased, fewer colonies or plaques are included in the count. As adjustments are made to the threshold, an onscreen display allows immediate feedback on the effects of altering the threshold value on the total colony or plaque count. Of the adjustable counting parameters, the setting for threshold level has the greatest effect on counting results.

The minimum size of a colony or plaque can also be defined prior to counting. This feature can be used to exclude small satellite colonies or contaminants from a count. If single colonies are being counted multiple times, the minimum colony size may need to be increased. If small colonies are unintentionally excluded from the count, the minimum colony size can be decreased.

Image Acquisition

In order to ensure the highest accuracy of counting, special care should be taken during image acquisition to maximize the contrast between colonies or plaques and their respective backgrounds. For white or blue bacterial colonies on an agar plate, white-light-box transillumination can be used to obtain images of adequate quality for analysis. However, for clear or blue plaques, standard white-light-box transillumination does not normally provide enough
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