Soil Ecology and Research Group
last update August 24, 2001
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Soil Ecology and Research Group last update August 24, 2001 |
Abstract
Europium staining, although a relatively new and largely unexplored procedure, may provide restorationists with a more accurate way to evaluate the number of bacteria and fungi in the soil. By using a specific chelate of the earth europium and a computerized image analysis program, we have been able to attain levels of information unattainable with other fluorescent microscopy techniques. While those techniques have often been unsuccessful and grossly inaccurate, europium staining provides 1) an ability to rapidly and accurately distinguish between viable cells and nonviable cells, organic matter, or inorganic particles; 2) a means to compare on-site microbial activity including the amount of disturbance and degradation to below-ground ecosystems; and 3) a way to establish a permanent data base for future study.
The technique is possible because organic compounds of some europium chelates absorb ultraviolet light and then transfer the energy to the europium ion, causing it to fluoresce (Scaff et al., 1969). Researchers have known for some time that the europium chelate, Europium (III) thenoyltrifuoroacetonate, stains viable cells and that a 50-percent solution of ethanol will wash away any background fluorescence (Scaff et al., 1969; Anderson and Westmoreland, 1971). Anderson and Westmoreland (1971) describe a procedure that creates a differential fluorescent stain (DFS) by mixing the chelate with a fluorescent brightener in 50-percent ethanol and water solution. The DFS-stained cells will emit a wavelength from 615 to 630 nanometers, which can be detected as red fluorescence.
We are using a procedure for europium staining outlined more recently by Jim Trent (1993). His research indicated that because of rapid decay times, samples must be refrigerated during transport and analyzed quickly once in the laboratory. To stain fungi, a lab worker pipettes 200 ml of the original soil mix (fresh soil and sterile water) into an empty, sterile test tube and then adds 1 ml of the DFS. They shake the tube gently by hand to mix the contents and set it aside for one hour to allow the stain to penetrate. The sample is then rinsed with a 50-percent ethanol wash. We use dilutions to make smears for bacterial staining. After swirling the dilution to get nonmotile bacteria into solution, the worker pipettes 8 ml of each dilution onto the appropriate slide. Smears are covered with the DFS and after one hour rinsed with 50-percent ethanol.
We then observe the stained slides on a microscope using an ultraviolet lamp as a light source. The microscope is equipped with a camera that sends images to a computer. We analyze the images by using the Imageviewer computer program, written for the Macintosh. A staff member or student magnifies the selected object and, using a histogram of intensity prepared by the computer, obtains a recordable pixel number (Hi-Lo area). The pixel number represents the area of the fungal hyphae or bacterium in the picture. Pixel numbers are obtained for each image, and for each separate object in the image. We can tabulate the data for each slide and calculate the total pixel areas. The totals for each slide are used to determine hyphal lengths (m/gram soil) and number of bacteria/gram soil.
Preliminary studies have shown the effectiveness of the europium staining procedure (Conners et al., 1993). For example, staining of disturbed and undisturbed soils at Red Rock Canyon State Park showed that undisturbed soils had greater hyphal lengths and larger bacterial populations. Moreover, it allowed us to determine that after rainfall on the site, these increased in undisturbed soils but remained constant in disturbed soils.
Although still experimental, it appears to us that europium staining is an excellent way to evaluate microbial populations in soils, and may be an excellent method for assessing disturbance and recovery. This procedure may also prove useful for other biological evaluations, including testing seed viability.
