Petri dish preparation

Simplicity of enumeration by image analysis greatly benefits from careful preparation of the Petri dishes. Effects caused by bubbles and foreign particles in the nutrient can, if at all, only be compensated with substantial effort in the processing of the image. One should also aim at constant plate quality, i.e., identical amounts of nutrient and constant layer thickness by precise horizontal alignment when pouring the dishes.

Colony clusters on the rim of the Petri dish are a substantial obstacle for automatic counting (true for manual counting as well). Various, in some cases quite complex algorithms have been published for treating clusters. Since we intended to develop a simple method it appeared to be more worthwhile to try and avoid clusters in the first place by choosing an appropriate inoculation method.

For this purpose lines drawn on the backside of each Petri dish (see Figs ​Figs11 and ​and2)2) indicate borders, which are not to be crossed during inoculation. If this is duly observed (e.g., with the help of a jig), colonies only grow within the outlined areas. These can subsequently be processed as separate enumeration areas by the image analysis software by defining respective regions of interest (ROIs). The lines facilitate alignment of the dish prior to image recording, making sure that inoculated areas are located within the ROIs of the processing software. In radial direction these regions end a few millimeters short of the rim of the Petri dish, preventing the formation of colony clusters in this area. Avoiding the dish edges also means avoiding the possibly thicker broth layer around the rim of the plate, thus achieving a more uniform background brightness of the image.