Materials and methods:

Soils

We used sterilized compost to avoid any effect of soil fungistasis and inoculated the fungi from pre-cultures on malt extract agar plates. Incubation was done in petridisks at room temperature.

Fluorochroming:

For this study, we used two stains, as follows.

Fluorescein-di-acetate (FDA):
This substance itself does not give any fluorescence; rather it is taken up by actively metabolizing cells or hyphae, where it is hydrolyzed enzymatically to yield free fluoresceine, a true fluorochrome.  Thus only living and actively metabolizing microorganisms become visible when stained with FDA (SÖDERSTRÖM & ERLAND 1986; CORREN et al. 1986; JENSEN 1994).

For our experiments a stock solution was prepared from 500 mg FDA dissolved in 100 ml acetone and stored at -18 ° C.  A working solution was prepared by diluting 0.1 ml of the stock solution in 5 ml 60 mM phosphate buffer pH 6.88. Since it degraded very rapidly, the working solution was freshly prepared for every observation.  This solution was mixed into the soil and microscopic observation started immediately; images were visible one minute after preparation.  Due to the rapid photofading of fluoresceine, staining lasted not longer than 20 minutes.

Calcofluor White:
This stain (correct name "Calcofluor white M2R new") is an optical brightener with a high affinity to B-1,4-glucans such cellulose or chitin. It is thus specific for hyphal walls, it does not differentiate between living and dead tissue.  Calcofluor stains many types of fungal structures, for example spores or fruiting body initials (VON SENGBUSCH 1983; COHEN et al. 1987). A stock solution was prepared from 50 mg calcofluor dissolved in 5 ml buffer (TRIS pH 9,0). The working solution consisted of 0.1 ml of stock solution in 9.9 ml buffer.  Both solutions are stable and were stored in the dark at room temperature. For microscopic observations, the working solution was simply mixed with the soil to be investigated immediately prior to observation.  The fluorescence was very stable, for example, mounts kept in a wet chamber could be studied for at least 24 h after staining. 

Treatment of soil samples:

In order to preserve the exact localization of hyphae, traps, or other specific structures and surrounding soil particles, the soil samples had to be treated very carefully. Soil samples were cut vertical from the surface to the ground of the petridisk with cover slides to get a number of soil slides with a depth of 2.5mm in average.  Each soil slide was placed on one cover slide. Staining and manipulation were done with much care to avoid distortion of the native structure. Observation was best through the slide in the hanging drop technique (JENSEN 1994; JENSEN & LYSEK 1995).

Microscopic and photographic equipment:

Observations were made using a Leitz Dialux 20 research microscope equipped with an (epi-)fluorescence illuminator PLOEMOPAK 2.4 supplied with a mercury lamp HBO 50 W (Osram).  The filter blocks used were A, E3, I3 and N2 (JENSEN 1994).  Photographs were taken with a MPS 11 microscope camera connected with a MPS 15 semiphotomat (Wild). Kodak negative films of 100, 200, 400 and 1000 ASA were used.

SEM-photos were taken using a Cambridge stereoscan 90 B 100/SE scanning electron microscope; for details see NEUMEISTER (1994).

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