Measurement of bacterial size via image analysis of epifluorescence preparations: description of an inexpensive system and solutions to some of the most common problems

SUMMARY: Computerized image-analysis of epifluorescence preparations is the most accurate and simple method for the estimation of bacterial size. We present a simple and inexpensive image-analysis system used to measure and count planktonic bacteria and presently in operation in our laboratory. We show that there is a wide range of image exposures (brightness) over which the system performs correctly. Even though the procedure involves some steps that depend upon operator intervention, the results obtained are highly reproducible and we have estimated the among-operator variability at 5%. We then discuss the advantages and disadvantages of different algorithms used for the estimation of volume from two-dimensional images and we identify those that perform better for curved and unusual cells. We finally estimate that 4 to 6 images and 200 - 250 cells are the optimal number of images to be processed and cells to be measured to obtain accurate estimates of population values with the minimum effort. These calibrations should be useful to all those laboratories that are implementing image-analysis systems.

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