Comparison between fluorescein diacetate and differential fluorescent staining procedures for determining fungal biomass in soils

Abstract

Soil microbial biomass is often used as an indicator of the state of processes and fertility in soil, yet most currently available techniques determine the total hyphal length (active live + inactive live + dead), rather than viable (active + dormant) or metabolically active portions of the hyphae. Inability to determine which pool is being measured can result in large errors in developing models of fungal community dynamics. The development of a quantitative relationship between active fungal biomass and viable hyphae needs to be developed in order to produce accurate measures of below-ground processes. Fluorescein diacetate (FDA) determines the active portion of the fungal community in soil while differential fluorescent stain (DFS) is a putative vital stain. We compared the amount of fungal biomass determined using FDA and using DFS, in three soils from southern California to determine the degree the to which the pools determined by the two stains is correlated. FDA and DFS measured different amounts of hyphae in each soil type sampled, yet the two techniques measured the fungal biomass in the same order of magnitude at all sites, indicating that DFS is most probably a measure of the active fungal biomass in soil. While there were several possible explanations for the differences in the amount of hyphae detected by the two stains, the most probable is that there are differences in the physical location of the stains within the hyphae, as shown by staining pure cultures of fungi. The large effects of storage on the fungal biomass detected in this study indicate that soil samples must be sampled rapidly after removal from the field. These results suggest that DFS is an activity stain that is more suitable for large sample numbers, in that prepared slides can be stored and analyzed microscopically at a later date.