ImageJ is a freeware image analysis program that can be used for many image processing and analysis. To save time and prevent inconsistencies, a number of image processing software programs, such as ImageJ, have been developed. However, the manual counting of colonies is time-consuming and imprecise. Of these methods, the agar plate method is commonly used to assay the survival rate of microbes. All rights reserved.Īnd form colonies on the plate, by counting the number of colonies, the number of viable bacteria can be obtained. The agar plate method (Barbosa, 1995) involves smearing the diluted bacterial suspension on an appropriate culture medium. The total number of bacteria in the original sample can then be calculated according to the number of colonies that form on the membrane filter. The microorganisms remain on the membrane, which is then placed on a culture medium. The membrane filter method (Inatomi, 2003) involves passing suitably diluted samples through a membrane filter with pore diameters smaller than those of the microorganisms. The amount of light that passes through the suspension is indicative of the concentration of certain bacteria that do not allow light to pass through. Spectrophotometry (Schmidt and Schmidt, 2004) is a quantitative measurement of the optical transmission of a bacterial suspension as a function of wavelength. Filters of various wavelengths convert these signals into electronic signals to enable the counting of bacteria. Bacteria are placed in a flow cytometer, in which the fluorescent substances they carry are excited by lasers set to particular frequencies for the generation of optical signals. Flow cytometry (Macey, 2007) combines the use of bacterial properties with various fluorescent substances. This usually involves counting the number of bacteria in a unit volume of bacterial broth using various methods, including flow cytometry, spectrophotometry, membrane filtering, and the agar plate method. The selection of antibiotics (Van Doorn et al., 2000), toxicology tests (Chen et al., 2004), and the evaluation of food and drug safety (Itoh et al., 1998) require the determination of microorganism survival rates to verify research achievements. Introduction Bacterial growth is an essential indicator in many studies on microorganisms. A user-friendly graphical user interface is also developed and freely available for download, providing researchers in biomedicine with a more convenient instrument for the enumeration of bacterial colonies. Comparisons show that the proposed system is an effective method with excellent accuracy with mean value of absolute percentage error of 3.37%. The performance of the proposed system is compared with verified manual counts, as well as with two freely available counting software programs. To accurately estimate the number of viable bacteria in a known volume of suspension, colonies distributing over the whole surface area of a plate, including the central and rim areas of a Petri dish are taken into account. To save time and prevent inconsistencies, this study proposes a fully automated counting system using image processing methods. The number of colonies in a culture is counted to calculate the concentration of bacteria in the original broth however, manual counting can be time-consuming and imprecise. Journal of Microbiological Methods journal homepage: Automated counting of bacterial colonies by image analysis Pei-Ju Chiang a,b, Min-Jen Tseng c, Zong-Sian He a,b, Chia-Hsun Li a,b a b cĭepartment of Mechanical Engineering, National Chung Cheng University, Chia-Yi, Taiwan Advanced Institute of Manufacturing with High-Tech Innovations, National Chung Cheng University, Chia-Yi, Taiwan Department of Life Science, National Chung Cheng University, Chia-Yi, TaiwanĪrticle history: Received 4 August 2014 Received in revised form 14 November 2014 Accepted 14 November 2014 Available online 21 November 2014 Keywords: Automated colony counting Image analysis Petri dishĪ b s t r a c t Research on microorganisms often involves culturing as a means to determine the survival and proliferation of bacteria. ![]() Journal of Microbiological Methods 108 (2015) 74–82Ĭontents lists available at ScienceDirect
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