Effects of Sediments on Estimates of Bacterial Density

We compared direct epifluorescent microscopic bacterial counts using either acridine orange [AO; 3,6-bis(dimethylamino)acridinium chloride] or DAPI (4',6-diamidino2-phenylindole) in the presence and absence of fine sediments. Differences in a time-series of population estimates obtained using either AO or DAPI direct-count methods were greater in sediment-amended cultures. In the presence of sediments, DAPI yielded significantly lower estimates of bacterial densities than AO. A sediment-addition experiment indicated that DAPI counts may be more sensitive to sediment presence than AO counts, although interaction between treatments (stain, sediment) clouds statistical interpretation. Variation between replicate cultures was greater than between subsamples of preserved combined samples. Mean differences attributable to choice of stain may be as great as differences attributable to potential masking effects of sediments. Despite culture variability, DAPI and AO methods yielded count estimates agreeing within an order of magnitude. Greater correspondence between counts was observed for turbid natural water samples spanning a more limited range of bacterial concentrations. Identifying and interpreting the biotic and abiotic factors responsible for the regulation of bacterial numbers is a major goal of microbial ecology. Bacteria have been enumerated in several ways, with many of the same techniques being applied in a variety of systems. If reported usage is indicative, methods of direct microscopic visualization are increasingly favored over standard platecount techniques which, by necessity, exert a selective effect leading to underestimates of total bacterial numbers. A serious and recognized drawback of plate counts is that no single medium will culture all bacteria (Buck, 1979), yet the presence of large numbers of bacteria (regardless of culturability) may be of great significance. Recently, epifluorescent direct-count techniques have become the methods of choice for enumeration of total bacteria (Fry, 1988). However, clear differences in numbers of bacteria observed depend upon both staining technique and physico-chemical characteristics of the sample. Two fluorochrome stains are most often used in direct-count methods. DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific stain that fluoresces blue or bluish-white when bound to DNA and excited with light at a wavelength of 365 nm. When unbound or bound to non-DNA material, it may fluoresce over a range of yellow colors. As with the other most commonly used strain, AO [3,6-bis(dimethylamino)acridinium chloride or acridine orange], bacteria are identified based not only upon color but also upon size and shape. AO binds to both DNA and RNA, and the excitation maximum for AO is approximately 470 nm. Both DAPI and AO stain bacteria and other fine particulate organic matter (FPOM) differentially. Two experiments were performed to assess differences in bacterial directcount methods. In particular, we were interested in potential differences in counts obtained using DAPI and AO. At the same time, we examined differences TRANS. AM. MICROSC. SOC., 112(4): 316-330. 1993. ? Copyright, 1993, by the American Microscopical Society, Inc. This content downloaded from 157.55.39.163 on Wed, 21 Sep 2016 05:28:09 UTC All use subject to http://about.jstor.org/terms VOL. 112, NO. 4, OCTOBER 1993 in our ability to enumerate bacteria in the presence or absence of fine inorganic sediments (primarily silts and clays) and whether this ability was influenced by choice of stain. Environmental samples also were collected and bacterial counts obtained using the two stains were compared. Particle-attached bacteria pose special problems because of masking effects and the need for dilution of high-turbidity samples. Turbid samples are encountered frequently in many types of studies (e.g., studies examining stormflow, estuarine or saltmarsh water, pore water, sediments, and soil water). The frequent occurrence of sediment-laden samples and common usage of DAPI and AO direct-count (AODC) methods in the enumeration of bacteria have created the need for further methodological comparisons of the type presented here. MATERIALS AND METHODS