Mounted(euparal or other suitable mountant).

Thus a soil-agar film of

known thickness (0.1 mm, the conventional depth of the well of a
hemocytometer slide)is mounted on a microscope slide. The prepared film
is observed under the microscope at known magnification (400-1000x)
and hyphal lengths in each of a number of random fields are assessed
using the intersect method (alson 1950). Replicate observations of each of
a number of replicate soil films are assetial (Frankland et al. 1978). One of
the problems of this method is the difficulty of distinguishing between live
and dead hyphae in the agar films. The use of fluorescent dyes with agar
fims is impratical, but observation of films stained with phenilic aniline
blue has allowed estimations of live (stained) and dead (unstained)
hyphae. Obserfation of films under phase contrast microscopy allows
distinctions between hyphae with cell contents and empty hyphae
(frankland 1975, Visser and parkinson 1975).
Knowing the length of hyphae per microscope field, the area of the
field, the depth of the film, and the dilution factor in preparing the soilwater-agar suspension, the length of hyphae per unit weight of soil can be
calculated. From these data hyphal standing croup ((dry weight) can be
calculated given data on the average radius of observed hyphae, an
average spesifit gravity of hyphae, and an average value for hyphal
moisture content (Parkinson et al. 1971, Frankland et al. 1978)
Membrane Filter Method. In an attempt to reduce the perceived
tediousness of the soil-agar film method and to allow the use of
fluorescent staining, Hansen et al. (1974) developed a membrane

filtration method. In this method 0.5-1.0 g of soil is suspended in 500 ml of

water, homogenized (30-50 seconds, depending on soil type) in a waring
blender, and known volumes of the suspension are membrane filtered
(0.45 m Oxoid standard grade). Eac filter is placed on a slide, allowed to
dry, and cleared (liquid paraffin or decahydronaphthalene for Oxoid
filters). If fluorescence microscopy using incident light is available,
clearing is unnecessary. The filters are observed and hyhal lengths
recorded (as described for the soil-agar film method).
Paul and Johnson (1977) described a variation of this method where
filters are stained with water-soluble aniline blue (1% w/v in 0.2 M K 2HPO4,
pH 8.0), the filters being observed using bright-field

illumination.

Soderstrom (1977) has described a vital method (fluorescein diacetate) in

an attempt to distinguish clearly between live and dead hyphae.
Assessment of these methods is given in Parkinson (1986).
Pure Culturing of Fungal Isolates and Their Preser vation
Detailed accounts of methods for obtaining pure cultures of fungal
isolates, of media on which fungi may be grown, and of methods for
presevation of fungal cultures