Page 38 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
WU095-02
Methods of polyphosphate assay
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The method used for determination of PolyP, which is based on the Mn -induced
2+
quenching of the fluorescence of the calcium indicator Fura-2, has been described (Lorenz
et al., 1997a). The effect of Mn 2+ ions on the Fura-2 fluorescence is gradually removed in
the presence of increasing PolyPs concentrations; this allows the quantification of PolyPs
isolated from tissues or cells. The described method has some advantages when compared
with the conventional detection procedures based on the metachromatic effect. It can be ap-
plied to the determination of pyrophosphate, tripolyphosphate and other short-chain PolyPs
not detectable by toluidine blue (Lorenz et al., 1997a).

2.4 Cytochemical Methods

The oldest and most extensively used method for determination of PolyPs in biological
materials, although of course the least accurate, is based on the staining of cells and tissues
by certain basic dyes such as toluidine blue, neutral red and methylene blue. The presence
of condensed phosphates in the organisms is judged by the appearance in the cells of
metachromatically stained granules, or volutin granules.
Two basic principles are involved in metachromasia: first, the interaction between dye
and substrate molecules, and secondly, the interaction between adjusted dye molecules
aggregated to the substrate. A striking change in the absorption spectrum of a metachromatic
dye in the presence of polyelectrolyte is generally characteristic of the specific nature of the
polymer. For example, such changes have been related to the chain length, conformation
and the functional group of an individual polymer. The interaction between the dye and the
polymer is influenced by experimental conditions such as pH, temperature, ionic strength,
and the molar ratio of the polymer residues to the dye molecules.
When ‘Loeffler’s methylene blue’ is used, the PolyP-containing granules appear pink–
violet on the blue background of the cells (Murray et al., 1994), while ‘Neisser staining
method’ gives purple–black granules on the yellowish-brown background of the counter-
stained cells (Bartholomew, 1981). Neisser staining is more suitable for determining PolyP
accumulation than Loeffler’s method because of its higher contrast between the granule
and the cell (Serafim et al., 2002). Toluidine blue, which shares the same metachromatic
properties as methylene blue, can also stain PolyP granules.
The staining by basic dyes such as methylene blue, toluidine blue and neutral red has
been used for the detection of PolyPs in living organisms for a long time (Wiame, 1946,
1947a,b, 1948, 1949, 1958; Macary, 1951; Widra, 1959; Drews, 1960 a,b; Ebel, 1952d; Ebel
et al., 1955, 1958a,b; Ebel and Mehr, 1957; Ebel and Muller, 1958; Tewari and Krishnan,
1959; Prokof’eva-Bel’govskaya and Kats, 1960; Dmitrieva and Bekker, 1962; Voelz et al.,
1966; Tijssen et al., 1982; Lopez-Revilla and Gomez-Dominiguez, 1985; Suresh et al.,
1985) and up to date has been one of the simplest and cheapest PolyP visualizing methods
(Rees et al., 1992; Leitao et al., 1995; Imsiecke et al., 1996; Serafim et al., 2002). PolyP
staining first showed the presence of characteristic granule clusters in activated sludge and
suggested the existence in them of PolyP-accumulating bacteria (Fuhs and Chen, 1975). The
staining method of PolyP detection is often used in the study of polyphosphate-accumulating
microorganisms of activated sludge (Suresh et al., 1985; Rees et al., 1992; Serafim et al.,
2002).