SOIL ORGANISMS - Soil Ecology - page 208

The soil suspension may be obtained by a gentle trituration in water, or a more intense
dispersion using a blender or ultrasound. The latter methods which disintegrate microag-
gregates generally give higher estimates (see, e.g., Babiuk and Paul, 1970).
Micro-organisms are observed in phase contrast or fluorescence microscopy, using
appropriate stains such as aniline blue and aceto-orcein for fungal hyphae and acridine
orange for bacteria. Stains may be used in combination as proposed by Anderson and
Westmoreland (1971) and fluorescent stains such as fluorescein isothiocyanate (FITC)
may also be used (Jenkinson and Ladd, 1981).
(c) Chemical methods include estimation of the soil concentrations of ATP
(Ausmus, 1973), D N A (Torsvik and Goksoyr, 1978), phospholipids (White, 1988;
Zelles et al. 1995), nucleic bases (Cortez and Schnitzer, 1979) or chitin, an important
component of hyphal membranes, and hexosamines (Swift, 1973).
(d) Physiological methods assess microbial enzymatic activity or respiratory
activity under different conditions.
Relating the activities of soil enzymes to microbial abundance has proved
difficult because a number of other soil characteristics also determine enzyme activity.
An unknown proportion of this activity may be attributed to extracellular enzymes fixed on clay
minerals (Burns, 1986). Schnürer and Rosswall (1982) proposed that the hydrolysis of fluores-
cein diacetate (FDA) into fluorescein is a useful index of microbial activity. Most soil micro-
organisms are able to effect this transformation and the method is therefore considered valuable.
(e) Respirometric methods are widely used. Direct respirometry is widely used for
comparative studies (see, e.g., Petersen's method as modified by Rashid and Schaefer,
1985; Verdier, 1983). Quantitative estimates may be derived by measuring the increased
respiration that occurs after adding glucose to the soil (Anderson and Domsch, 1978).
The use of selective respiratory inhibitors allows the separation of bacterial and fungal
respiration. However, their utilisation may be difficult.
(g) The biocidal method (Jenkinson and Powlson, 1976) is currently the common-
est technique used to estimate microbial biomass. The soil sample is fumigated with
chloroform to kill the microflora and is then incubated under controlled conditions.
Microbial biomass is estimated from the magnitude of the “flush” of carbon dioxide
produced from the decomposition of dead micro-organisms by surviving or newly-
inoculated ones. Microbial biomass estimated by this technique includes such soil
microfaunal components as protists and nematodes.
The biocidal method has been further developed and improved by a number of
authors. Specific methodologies have been designed to measure the amount of nitrogen
contained in biomass ( e.g., Brookes et al., 1985; Amato and Ladd, 1988; Alef et al.,
1988). A simplification of the method consists of an extraction and titration of microbial
organic C rendered extractable by 0.5 M (Vance et al., 1987). A similar method
has been proposed by Davidson et al. (1989) for measuring microbial biomass nitrogen.
Finally, Hendricks and Pascoe (1988) have demonstrated that micro-organisms may be
killed by microwave irradiation instead of chloroform with equivalent results.
(f) Comparison of methods. Several authors have compared the results obtained using
the different methods available.
Culture techniques produce the most variable results. Variability decreases with
direct counts and chemical methods and is least when physiological methods are used