INSITU ENUMERATION OF BACTERIAL ADHESION IN A PARALLEL PLATE FLOW CHAMBER - ELIMINATION OR IN FOCUS FLOWING BACTERIA FROM THE ANALYSIS

Automated in situ enumeration using image analysis of bacterial adhesion to solid substrata in, e.g., a parallel plate flow chamber requires sophisticated methods to ensure that in-focus flowing bacteria are separated from the adhering ones and eliminated from the analysis. In this paper, three automated methods to eliminate in-focus flowing bacteria from the analysis, based on one snapshot image, addition of snapshot images, and multiplication of snapshot images were compared with a manual counting method. All automated enumerations were done as a function of the black and white threshold installed to distinguish adhering bacteria from their background. Experiments were carried out on substrata with different transparency, yielding different illumination conditions, and at two flow rates, yielding different velocities of in-focus flowing bacteria. Results showed that the automated enumeration techniques described in this paper all yield counts that compare well with manual counts, even for the snapshot image, which does not involve any precaution to eliminate in-focus flowing bacteria from the analysis. A major advantage, however, of using the addition or multiplication of images described is that one can allow a 2-3-fold larger variation in threshold value than for enumeration based on a snapshot image and still get excellent counts. This makes the entire experimental procedure less critical. In addition, the allowable variation in threshold value is slightly larger for the multiplication procedure than for the addition procedure.