Selenium Uptake of Aeromonas Hydrophila as Monitored by Energy Dispersive Spectroscopy
IAAAM 1991
Bobby Bramlett; Nancy Smith; Beverly Dixon, PhD
Bobby Bramlett; Nancy Smith; Beverly Dixon, PhD


Studies in our laboratory have shown that selenium affects the growth rate of bacteria such as Aeromonas spp. and Salmonella spp. In this study, environmental isolates of Aeromonas hydtophila were grown in various concentrations of sodium selenite. Ultra structural examination of these cells revealed atypical morphology and electron-dense granules within the cytoplasm and cell membranes. An energy dispersive spectrometer combined with a scanning transmission electron microscope (STEM) was used to determine the localization and identity of selenium within the cells. The effect of selenium on antibiotic susceptibility is currently under investigation.


Selenium (Se) Ls a naturally occurring non-metallLc element that is ubiquitous throughout the environment. It is an essential micronutrient that plays an important role in many biological processes in plants as well as being involved Ln cellular repair mechanisms (Maters et al 1988) and immunological processes Ln animals. At low levels, selenium in combination with vitamin E has been shown to be an immunopotentiator (Blazer 1984). Also, it has been demonstrated that low levels of selenium reduce the toxic effects of cadmium and mercury. However, at slightly higher concentrations selenium is extremely toxic.

In its elemental state, selenium is insoluble in water and considered to have minimal toxicity. But, the two soluble forms selenate (+VI) and selenite (+IV) are considered to be extremely toxic. As a result of industrial and agricultural processes, the two soluble forms of selenium (selenate and selenite) are starting to accumulate in lakes, rivers and estuaries throughout the central California valley. Because of these continuously increasing levels of selenium in these areas, aquatic animals are continuously being exposed to abnormally high levels of selenium which intern may produce toxic effects. These effects may range from physical malformations during embryonic development to sterility and death.

In recent years, it has been necessary to investigate the possibility of a treatment system to remove the soluble forms of selenium from areas which are considered to contain unusually high levels of toxic selenium. A number of microorganisms are capable of reducing soluble selenium to its elemental state (Macy 1989). The potential exists for a treatment system that uses indigenous bacteria in a treatment system to remove toxic selenium by allowing microorganisms to reduce selenate and selenite to its nontoxic elemental form (Maters 1989). In order for this type of treatment to be realistic utilized, it is necessary to demonstrate that selenate and selenite are actually being converted to elemental selenium by the microbes.

Materials and Methods

Environmental isolates of Aeromonas hydrophila were grown on agar plates containing 0, 10, 50, and 100 mg/1 (ppm) of sodium selenite for 24 and 48 hours at 25°C. Colonies were removed from the plates and fixed in 2.5% glutaraldehyde in 0.1M Na-cacodylate buffer, pH 7.2, for 1 hour. Following fixation, specimens were pelleted and rinsed with 0.1M Na-cacodylate buffer followed by dehydration through a graded series of ethanol. Dehydrated specimens were embedded in L.R. White resin. A portion of the sample grown in 100 mg/1 selenite was used to isolate granules. Granules were isolated by subjecting the cells to distilled water followed by a regime of detergent and bleach to remove adhering cellular debris. The isolated granules were deposited on formvar coated copper grids for analysis. All samples were examined with a JEOL 200 CX scanning transmission electron microscope (STEM) equipped with a Kevex analyst 8,000 EDS microanalysis system.


Electron dense granules observed within the bacteria were most often localized at the cell periphery and frequently could be seen protruding from the cell. In addition, it was not uncommon to see granules outside the cells. The extra cellular granules appeared to be surrounded by membranes. X-ray analysis of the isolated granules and intra- and extra cellular densities show only selenium peaks (and copper grid). Further analysis using energy loss spectroscopy and micro diffraction methods substantiate that the densities are elemental, amorphous selenium.


As a result of industrial and agricultural processes, wetlands are accumulating high levels of selenate and selenite. As a result, aquatic animals are increasingly exposed to the toxic forms of selenium. The importance of bioaccumulation of selenium in the environment is just beginning to emerge. It is evident that soluble selenium is becoming an increasing problem and it must be dealt with in some way. Data suggests that indigenous bacteria are capable of converting selenate and selenite to its non-toxic elemental form.


1.  Blazer, V.S. and Wolke, R.E. The effect of a-Tocopherol onthe Immune Response and Non-specific Resistance of Rainbow Trout. Aquaculture 37,pp. 1-9, 1984.

2.  Davis, E.A., Maier, K.J., and Knight, A.W. The Biological Consequences of Selenium in Aquatic Ecosystems. Calif. Agricult. 42,pp.18-20, 1988.

3.  Macy, J.M. et al., FEMS Microbiol. Lett., 52 195, 1989.

4.  Maiers D.T. et al., App. Environ. Micro. 54, 2591, 1988.

Speaker Information
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Bobby Bramlett

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