An experimental setup and segmentation method for CFU counting on an agar plate for the assessment of drinking water

Abstract

Quantification of bacterial colonies on an agar plate is a daily routine for a microbiologist to determine the number of viable microorganisms in the sample. In general, microbiologists perform a visual assessment of bacterial colonies which is time-consuming (takes 2 min per plate), tedious, and subjective. Some automatic counting algorithms are developed that save labor and time, but their results are affected by the non-illumination on an agar plate. To improve this, the present manuscript aims to develop an inexpensive and efficient device to acquire S.aureus images via an automatic counting method using image processing techniques under real laboratory conditions. The proposed method (P_ColonyCount) includes the region of interest extraction and color space transformation followed by filtering, thresholding, morphological operation, distance transform, and watershed technique for the quantification of isolated and overlapping colonies. The present work also shows a comparative study on grayscale, K, and green channels by applying different filter and thresholding techniques on 42 images. The results of all channels were compared with the score provided by the expert (manual count). Out of all the proposed method (P_ColonyCount), the K channel gives the best outcome in comparison with the other two channels (grayscale and green) in terms of precision, recall, and F-measure which are 0.99, 0.99, and 0.99 (2 h), 0.98, 0.99, and 0.98 (4 h), and 0.98, 0.98, 0.98 (6 h) respectively. The execution time of the manual and the proposed method (P_ColonyCount) for 42 images ranges from 19 to 113 s and 15 to 31 s respectively. Apart from this, a user-friendly graphical user interface is also developed for the convenient enumeration of colonies without any expert knowledge/training. The developed imaging device will be useful for researchers and teaching lab settings.