A plate count method for aerobic cellulose decomposers in soil by Congo red staining

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Abstract
The ability of microorganisms to decompose polysaccharides such as cellulose is of considerable importance in terms of the carbon cycle in soil ecosystems. In order to analyze the behavior of cellulose decomposers in soil, a quantitative estimation is indispensable. Someya (1980) modified the plate count method for the enumeration of cellulose decomposers in soil developed by Hankin and Anagnostakis (1977). Carboxymethylcellulose sodium salt (CMC) is used as the substrate which, after incubation, is precipitated with cetyltrimethylammonium bromide (CTAB, 10 g L-1) to visualize the zone of hydrolysis (clear zone) on an agar plate. The method has been widely used for the enumeration of cellulose decomposers in soil. However, one of the shortcomings of the method is that, with the time of incubation, the large clear zones formed by rapidly growing cellulose decomposers overlap with each other and/or mask the small clear zones formed by slower or weaker cellulose decomposers (Someya 1980). Although such overlapping can be prevented to some extent by increasing the concentration of CMC and agar in the medium, the hydrolysis zones are too blurred for counting (Someya and Furusaka, 1981). In addition, the enumeration must be performed rapidly since some zones appear clearly for only a few minutes (Someya 1992). On the other hand, Teather and Wood (1982) used Congo red (CR) as a stain for residual CMC for counting anaerobic cellulolytic bacteria in bovine rumen. However, the CMC concentrations they used (0.5-1 g L-1) were too low to count aerobic cellulose decomposers in soil. In this paper, a method in which a higher concentration of CMC was used and the CR staining technique was applied was adopted for counting aerobic cellulose decomposers in soil.

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