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J Microbiol Methods. 2000 Feb;39(3):213-24.

Comparative study of the abundance of various bacterial morphotypes in an eutrophic freshwater environment determined by AODC and TEM.

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  • 1Institut für Medizinische Biologie, Abtgr. Allgemeine Mikrobiologie, Universität Wien, Austria.


Transmission electron microscopy (TEM) and epifluorescence microscopy were used to obtain comparative measurements of total bacterial counts, and to enumerate abundances of various bacterial morphotypes in an eutrophic freshwater habitat. Although particulate matter would have been expected to interfere with counting by obscuring large areas of the electron microscope grids, estimates of total bacterial abundance made by TEM were on average 1.2 times greater than those obtained using the acridine orange direct counting method (AODC). However, the precision of the AODC method was greater than that for TEM, with a coefficient of variation (C.V.) of 4.0% versus 8.8%, respectively. The total bacterial abundance ranged from 1.1 to 3.2 x 10(6) ml(-1). As was the case for total bacterial density, the numbers of rod- and vibrio-shaped cells were lower when counted in the epifluorescence microscope, indicating the presence of potential starvation forms or ultramicrobacteria. Greatest variations in counts made by TEM and AODC were found for filamentous and coccoid bacteria. Counts of filamentous bacteria made by AODC were only about half of those detected by TEM. In contrast, cocci were on average 1.5 times greater when counted by AODC compared to TEM estimates. Both counting differences were probably caused by the morphology and low density of filamentous and coccoid bacteria (1.7 and 1.4 x 10(5) ml(-1), respectively), which led to an uneven distribution on polycarbonate filters as well as on electron microscope grids. Besides, cocci might easily be mistaken for large viral particles when counted by AODC. Hence, the study supports the use of TEM over AODC for obtaining accurate estimates of total bacterial abundance and especially bacterial morphotypes in natural waters.

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