Page 40 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
WU095-02
Methods of polyphosphate assay
24 March 9, 2004 15:25 Char Count= 0
DAPI staining was used for identification of vacuolar PolyPs (Allan and Miller, 1980) and
cell-surface PolyPs (Tijssen et al., 1982) of yeast. This method is commonly used in the iden-
tification of PolyP-accumulating microorganisms from activated sludge during the study of
Enhanced Biological Phosphorus Removal (EBPR) (Serafim et al., 2002). The DAPI–PolyP
staining has often been used in studies of EBPR because of the possibility to combine this
procedure with in situ molecular identification (FISH analysis). In the latter analysis, the
16S rRNA fluorescent probes specifically bind with the target bacteria (Wagner, et al., 1994;
Bond and Rees, 1999; Bond et al., 1999; Kawaharasaki et al., 1999; Crocetti et al., 2000).
They appear as fluorescent cells, and bacteria belonging to a specific taxonomic group may
be identified in mixed biomass such as activated sludge. Procedures that combine FISH with
methylene blue staining (Crocetti et al., 2000) or with DAPI staining (Kawaharasaki et al.,
1999) allow visualization of PolyP granules in taxonomicaly identified cells. The sequential
FISH, DAPI and polyhydroxyalcanoates (PHAs) staining methods have been described (Liu
et al., 2001). It should be noted that further studies of various samples and ad-
equate conditions are required to check the reliability of these new cytochemical
approaches.

2.5 X-Ray Energy Dispersive Analysis

PolyP-containing deposits in cells are also visualized by electron microscopy as electron-
dense regions, and when such microscopy is combined with X-ray energy dispersive analy-
sis, it is possible to detect the phosphorus, presumably present as PolyPs, and metal cations
such as Na, K, Ca, Mg, Mn, Zn, Ba and Al. Such analyses are useful to identify the metal
composition of PolyP granules and to obtain evidence of PolyP involvement in cation
chelation. In a number of studies, this method was successfully used for the detection and
chemical analysis of PolyP granules in various organisms (Ashford et al., 1975; Callow
et al., 1978; Adamec et al., 1979; Doonan et al., 1979; Baxter and Jensen, 1980a; Scherer
and Bochem, 1983; Vo˘r´ı˘sek and Zahleder, 1984; Pettersson et al., 1985; Ogawa and Amano,
1987; V¨are, 1990; Ashford et al., 1999; Ramesh et al., 2000; Schonborn et al., 2001).
Interesting data on the structure and formation of PolyP granules in cyanobacteria were
obtained by electron microscopic and cytochemical methods (Jensen, 1968, 1969; Jensen
and Sicko, 1974; Sicko-Goad et al., 1975; Jensen et al., 1982).
The study of microbial cell granules by X-ray dispersive analysis revealed that the com-
position of the granules changed markedly depending on the chemical and ionic composition
of the culture medium. For example, the quantitative ratios of Ca, Mg and K in PolyP gran-
ules of bacteria in wastewaters varied depending on the influence concentrations of these
metal cations (Schonborn et al., 2001). The quantitative X-ray analysis of laboratory grown
cyanobacterium Plectonema boryanum and bacterium Staphylococcus aureus revealed that
−8 −8
typical in vivo PolyP bodies contain (in µg): O (4.3 × 10 ), C (1.2 × 10 ), P (6.7 ×
−9
−9
10 ), Mg (1.3 × 10 ), Ca (6.7 × 10 −10 ), K (6.7 × 10 −10 ), Fe (6.0 × 10 −10 ), S (5.4 ×
10 −10 ) and Al (5.9 × 10 −10 ) (Goldberg et al., 2001).
This method has often been used in the investigation of PolyPs in mycorrhiza fungi
(Ashford et al., 1975; Callow et al., 1978; Orlovich and Ashford, 1993; Bucking et al.,
1998; Ashford et al., 1999). For example, an energy dispersive X-ray spectrum from a