S Y N G E N E
Application Note 07
A D IVISION OF THE SY NOPTICS GROUP
Colony and Cell Counting using GeneTools software Introduction GeneTools colony and cell counting software allows you to count bacterial colonies or plaques and cells present on a petri-dish or multi-well plate. The software will automatically count colonies, separating clusters so there is no need for manual intervention. Counting within a user definable frame, the results are fast and accurate. When using traditional colony counting software, the best results are obtained when the colonies are well separated on the plate. However, there are often a large number of colonies with many of those colonies overlapping. When using the more traditional software to count these types of plates the user has to manually manipulate the results to obtain accurate results. GeneTools software utilizes a separation technique when analyzing a colony image. The software initially counts all easily identifiable colonies then the software runs a proprietary separation algorithm before recounting the colonies to determine the final count result. To observe the separation of the colonies you can select ‘show the colony shapes ‘ on the image. To exclude regions of contaminated areas or bubbles in the medium you can draw around the region on the plate which you wish to be excluded from the count. Even using the most sophisticated algorithms, some colony plates make it difficult to obtain an automatic count that is as accurate as a manual one. This may be due to irregular shaped colonies, plate debris etc GeneTools software also allows the user to manually add and delete colonies once the automatic count has been performed. Simply check the box labelled Add/ remove colonies. Double click the left mouse button to add or remove colonies as desired. Use the zoom facility to view the colonies more clearly.
Figure 1: GeneTools analysis of an agar colony plate The blue squares present on the plate image show the counted colonies. The counted colony results are displayed in this figure in a table to the right of the plate image. Users can adjust the sensitivity to capture more or less colonies in the count according to their needs. The software allows colonies to be differentiated in area limits and size to ensure an accurate count of any plate.
Counting Coloured Colonies When working with chromogenic media and coloured colonies, GeneTools has a class split function, which enables automatic discrimination and counting of blue and white colonies on the same plate (Figure 2), a feature which many molecular biologists find very useful.
Counting from Agar Plates GeneTools has a built in function, which allows an automatic count of colonies from a framed circular image in seconds. Since counting is based on a userdefined, adjustable circle, an automatic colony or plaque count can be performed on any size of plate including standard 9cm plates and 15cm library plates this means that edge effects such as bubbles or pen markings can be excluded. (Figure 1).
Figure 2- GeneTools analysis of a two coloured agar colony plate The blue squares present on the image show the white colonies and the yellow circles show the blue counted colonies. The counted colony results are displayed in this figure in a table to the right of the plate image.
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S Y N G E N E
Application Note 07
A D IVISION OF THE SY NOPTICS GROUP
Determining Cell Numbers in Multi-well plates Utilizing the zoom function of a Syngene image capture system and GeneTools’ adjustable circular frame, users can obtain an image of cells grown in the well of a multi-well plate. GeneTools will then automatically quantify the amount of cell growth in the well in seconds and store the images of the plate well for future reference (Figure 3). This can be useful for microbiology techniques for studying the effectiveness of specific agents on the survival and proliferation of cells e.g. clonogenicity assays (Figure 4).
Incidence analysis GeneTools has a sophisticated spot analysis capability that can analyse microtitre plate wells. Images of microtitre plates are produced using Syngene’s microtitre prysm and this ensures images of clear circular wells within the plate’s image. GeneTools can then be programmed to find positive or negative results with parameters that are defined by the user. The results are displayed simultaneously in one window as a microplate image above an incidence table where positive and negative results are shown as a one or a zero respectively. Results from the spot analysis can be immediately transferred to Microsoft Excel spreadsheets or saved as text files for use in reports.
Conclusions
Figure 3- GeneTools analysis of cells growing in the wells of a multi-well plate The yellow circles present on the image show counted cells. The counted colony results are displayed in this figure in a table below the plate image.
The flexibility, GLP compliance and the availability of upgrades are all factors that potential users should consider when choosing image analysis software. GeneTools scores very highly in all of these areas. GeneTools is fully GLP compliant, with each analysed set of results being assigned a unique identity number so that even a re-analysed sample can be detected. In addition, Syngene provides all GeneTools software upgrades for life, free of charge to guarantee that GeneTools users will always have access to the most current application features. These benefits combined with Syngene’s expert advice, makes using GeneTools an extremely attractive option for many life science laboratories.
Syngene reserves the right to amend or change specifications without prior notice. This Application Note supersedes all earlier versions. All trademarks acknowledged. May 2010
Figure 4: A clonogenicity assay showing colonies stained with crystal violet This image was kindly provided by AstraZeneca, UK
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