Evaluation of DAPI direct count, computer assisted and plate count methods

Abstract

The feasibility of using automatic counting of bacteria stained with highly specific and sensitive fluorescing DNA stain DAPI, 4',6-diamidino-2-phenylindole, and direct manual counting to enumerate both pure culture of Pseudomonas putida overnight culture and sea water enhanced culture, was tested in correlation with plate direct counting, turbidity and absorbance at 600nm, to obtain cross validation. Six diluted samples from overnight pure culture of Pseudomonas putida and sea water culture were diluted and stained with DAPI. The images captured with epifluorescence microscope fit out with a digital camera, were counted automatically using Matlab software and manual counted. In addition, the turbidity and absorbance of both diluted samples was measured, as well as enumeration of colonies in diluted samples plated on the specific growth medium. The results indicated that automatic counting method could be employed successfully in the enumeration of bacteria, but with some limitations due to image analysis technique used and due to limitations of DAPI staining. Also the results indicated that absorbance and turbidity cannot be correlated with the number of bacteria in a sample.