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Counting Colonies or Plaques Directly from Images of Agar Plates

Novel capability of EagleSight software version 3.1

Michelle Cayouette Jane Moores Michael Steege Alan Orr
Stratagene Cloning Systems, Inc.

With the introduction of EagleSight software version 3.1, researchers have the ability to automatically count bacterial colonies and plaques on agar plates. Researchers can use the versatile features of the Colony/Plaque Counting function, such as flexible background-division settings, adjustable detection thresholds and user-definable constraints for minimum size of colonies and/or plaques, to obtain precise single- and two-color counts from various types of agar plates. In order to ensure accurate counting, images should be acquired in a manner that provides the best contrast between colonies or plaques and background. The image type should also be defined correctly prior to analysis.

The Eagle Eye II still video system is a digital imaging system used to document and analyze data from a variety of laboratory experiments. The systems software contains many tools that aid in acquiring and processing images.1,2 Calculations of molecular weight, integrated density and concentration can be made from images of stained nucleic acid or protein gels,3 chemiluminescent experiments4 or other applications. The analysis portion of EagleSight software version 3.1 has been enhanced with new colony- and plaque-counting capabilities. With these capabilities, researchers are able to count colonies and plaques directly from images of agar plates.

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The Colony/Plaque Counting function of EagleSight software version 3.1 is designed to provide quantitative information from images of plates quickly and easily. Either one or two color groups can be analyzed, which allows scientists using color-selection techniques to count both blue and white colonies (or blue and clear plaques) separately and concurrently (figure 1 and figure 2). In order to speed analysis, the counting region can be determined automatically based on sharp differences in pixel intensity, such as the differences that occur along the edges of a petri dish. Initially, counting begins automatically after the counting region has been delineated. If preferred, a region can be defined manually from an Advanced Options dialog box. Scientists also have the ability to exclude text written on plates without having to physically erase the text before imaging. Counting results can be modified by manually adding or removing individual colonies or plaques. During automatic colony or plaque counting, users should visually compare colonies or plaques present within the image of the plate to those being reported. If counted colonies or plaques do not correlate well with those on the image, counting parameters may need adjustment. Several adjustable counting parameters are provided to obtain accurate results for many types of bacterial or plaque plates.

Adjustable Counting Parameters Provide Flexibility and Accuracy

EagleSight softwares Colony/Plaque Counting function allows users to choose from several options for defining background. During counting, the colony or plaque vicinity is surveyed and an appropriate background value is assigned. In this way, each colony or plaque has a background value that corresponds to its location. The size of the colony or plaque vicinity is adjustable, providing the flexibility needed to accurately count colonies or plaques from images with 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 contrast between plaques and the bacterial lawn for precise analysis. In this case, an image with improved contrast can be acquired using overhead white-light illumination while the agar dish is resting on top of a piece of black, matte plastic. This method can also be used for acquiring images of small, abundant, white bacterial colonies on agar plates to increase contrast and enhance counting precision. Alternatively, plaque plates can be moved closer to the camera lens manually while using white-light-box transillumination. During all acquisition methods, the magnification should be adjusted so that the edges of the petri dish are fully visible and centered in the image. This adjustment will aid the automatic determination of the counting region, which is based on detecting the edge of the agar dish.

Defining Image Type

A critical factor in obtaining an accurate count is correctly defining the image type prior to analysis. When colonies or plaques appear lighter than the background of the plate, the image should be defined as white on black. Conversely, when colonies or plaques appear darker than the background of the plate, the image should be defined as black on white. If the image type is improperly defined, the count will not be accurate, and colonies or plaques that are counted will not correspond to those that appear on the image. When the Colony/Plaque Counting function is first enabled, an Image Type Selection dialog box is displayed, which prompts the user to specify image type. Before entering the Colony/Plaque Counting function, the image type can also be changed using the toolbar icon or the Analyze menu.


In addition to molecular weight and basic densitometry functions, the analysis portion of Eagle Sight software version 3.1 now includes a colony- and plaque-counting function. This function allows researchers to count colonies and plaques directly fro m images of agar plates. The Colony/Plaque Counting function is designed to quickly and easily provide quantitative information from either single-color or two-color colony and plaque plates. Adjustable detection-threshold levels, minimum size constraints and options for setting background levels enhance counting flexibility and increase accuracy of results.


The authors wish to thank Alexandra Bernardino, Marie Callahan, Wade Hareld, and Barbara Pagarigan for helpful discussions and product testing during development.


  1. Steege, M., and Cayouette, M. (1996) Strategies 9: 53-56.

  2. Cayouette, M., and Hansen, C. (1996) Strategies 9: 56-57.

  3. Cayouette, M., Steege, M., and Moores, J. (1997) Strategies 10: 21-23.

  4. Cayouette, M., and Moores, J. (1997) Strategies 10:56-57. THIS ISSUE.



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