Direct counting by epifluorescence microscopy is the best method available to determine total counts of aquatic bacteria. However, microscopic observation is tedious and time-consuming. A more rapid and certainly less subjective way of counting bacteria is to combine epifluorescence microscopy with an image analysis system. Surprisingly, although image analysis is now a relatively common method to measure the size of aquatic bacteria, very few studies have been devoted to the validation of total counts by image-analysis systems. In this paper, we present data on simultaneous determination of total counts of 4'6-diamidino-2-phenylindole (DAPI) stained bacteria by visual means and by image-analysed (Mudicam® system) epifluorescence microscopy methods.
The Olympus microscope BH2 is equipped for epifluorescence with a 100 W Hg lamp and a 100x oil immersion objective (Apo UVFL 160/1.3). The image analysis system consists et a high performance (5 x 10-4 lux) video camera (Lhesa LH40036) and an image processor which digitalizes the video image in a grey scale extending from 0 (black) to 255 (white) into a binary image with 512 x 512 pixels (8 bit, cyclope v 2.32, Digital Vision), and image analysis software (MUDICAM®. EAU). The samples were stained with DAPI (final concentration 2.5 µg/ml) and filtered through polycarbonate inters (0.22µm, Nuclepore Corporation). The surface area of the video image is 76 x 111 µm2.
The analysed samples come from culture collections of different bacterial strains (n = 30) submitted to different conditions and incubation times to obtain various physiological states (Table 1). The nature water samples were collected from several aquatic ecosystems : Rhône river, Mediterranean sea, Thau lagoon and Montpellier sewage waters (n = 50). The bacterial abundances ranged from 105 to 108 cells/ml and the size range of the cells varied from 0.63 to 17 µm2. Comparisons between the image analysis and visual counts were made on the basic of thirty fields per filter. The image analysis counts are based on a two step procedure. The video image of each microscopie field is first numerised and stored on a hard disk (153 Mo). When all the fields have been stored, the digitized images are submitted to an automatic thresholding which allows background substraction. Automatic counting of bacterial cells is then performed on the basis of object specifications defined by the operator. These specifications concern the minima and maxima values of the area (expressed in pixel numbers) and the fluorescence (expressed in gray levels) of the objects. The MUDICAM®EAU software also provides the mean number of cells per millilitre and the associated variance.
Average concentrations and confidence limits are shown in Table 2 for bacterial collection strain cultures and in Table 3 for water samples. When we compared visual and image analysis counts by- linear regression, the ability of the image analysis system to enumerate bacterial cells was clearly demonstrated. With bacterial culture (Fig. 2) and with water samples (Fig. 3), the coefficients of correlation were respectively r = 0.997 and r = 0.996 (p = 0.0001). The slopes of the models are not significantly different from unity and the Y-intercepts are not different from zero. Moreover we have compared the total visual counts of two experimenters and the image-analysed counts on eighteen random samples (Table 4). The variance analysis shows that there is no difference between the three methods, with mean value of 6.09, 6.08 and 6.11 for the image-analysed method, experimenter n° 1 and experimenter n° 2, respectively. While non significant, the greatest difference in counts was obtained between the two experimenters.
If may be concluded that the image analyser tested for total counts by epifluorescence microscopy is a precise and rapid procedure for the determination of total bacterial counts. This method may be standardized and its automation allows the analysis of many samples, an important advantage in ecological studies. Storage of the samples also allows one to treat a posteriori some complementary aspects of the total count, such as the double staining of bacteria. The image analyser tested is appropriate for bacterial ecology studies which require epifluorescence microscopy.
Bacterial count, epifluorescence microscopy, image analysis, bacterial collection strains, aquatic environmental bacteria, DAPI.
Got, P., Laboratoire d`Rydrobiologie Marine, U.R.A. CNRS 1355, Université Montpellier II 34095 Montpellier cedex 5