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materials. This limits the value of chitin assays except
in well-defined model systems.
An alternative method is to estimate theergosterol
contentby extracting ergosterol from a sample and
assaying it by high performance liquid chromato-
graphy (HPLC) and UV detection at 280 nm. Ergosterol
is the characteristic sterol of most fungal membranes
and is absent from plants and other soil organisms.
However, again the ergosterol content can vary
markedly between fungal taxa. In a critical analysis
of all such methods, including the assay of other “sig-
nature molecules” such as phospholipid fatty acids,
Olsson et al. (2003) concluded that the comparison of
biomass between different fungi is very difficult using
any currently available biochemical marker.

Antibody techniques

Fluorescent antibody techniques enable fungi to be
identified on the basis of their surface or internal anti-
gens. Antibodies are raised to the antigens of a specific
fungus by standard techniques, involving the inocula-
tion of rabbits, then the antibodies are used to detect
the fungus in a natural sample. For this, a secondary

antibody is used which binds to the primary antibody

and which is also tagged with a fluorescent compound

so that the target fungus can be seen under a fluores-

cence microscope. Alternatively, the secondary antibody

can be linked to an enzyme which releases a dye from

a colored substrate. This technique, termed ELISA

(enzyme-linked immunosorbent assay), can be used

to quantify the amount of specific antigen in a sample.

The main problem with both techniques is that the

antibodies can cross-react with the antigens of other

fungi. Monoclonal antibodiesoffer more specificity

because each is derived from a single cell line produc-

ing one specific antibody. An example of their use in

a different context was discussed in Chapter 10 (see

Fig. 10.16).

Fluorescent vital dyes

Fungi can be visualized directly by treatment with

fluorescent dyes (fluorochromes) that bind to the cell

surface or accumulate in specific cellular organelles. The

cells are then viewed under a fluorescence microscope

(Fig. 11.3). Some of these dyes, when pre-loaded into

hyphae, can be translocated for up to 9 mm as the

FUNGAL ECOLOGY: SAPROTROPHS 215

Fig. 11.3Examples of some fluorescent vital dyes; see color images on the Internet for higher resolution. (a) Hyphae
of Sclerotium cepivorumtreated with CMFDA, which accumulates in fungal vacuoles and shows as bright green fluores-
cence. (b) Hyphae and spores of Pythium oligandrum, stained with Cellufluorwhich binds to chitin in fungal walls
and fluoresces blue. (c) Hyphae of Fusarium oxysporumstained with DiIand DiO, giving green or red fluorescence of
the fungal membranes. (d) Hyphae of Botrytis cinereastained with CMAC, giving blue fluorescence of the vacuoles.
(e) Hyphae of Fusarium oxysporumstained with Nile red, showing intense yellow fluorescence of lipid droplets, while
the cytoplasm fluoresces orange.