Symbiotic Fungi: Principles and Practice (Soil Biology)

Chapter 6
Assessment of Phosphatase Activity
Associated with Mycorrhizal Fungi
by Epi-Fluorescent Microscopy
Ingrid M. van Aarle
6.1 Introduction
The use of the ELF-97 endogenous phosphatase detection kit, a fluorescence-based
method from Molecular Probes (Leiden, the Netherlands) for the assessment of
phosphatase activity associated with mycorrhizal fungi is detailed. The kit consists
of an ELF-97 phosphatase substrate [2-(5-chloro-2-phosphoryloxyphenyl)-
6-chloro-4-(3H)quinazolinone (CPPCQ)], an alkaline detection buffer and a mounting
medium, enabling the detection of alkaline phosphatase activity. However, use
of the substrate in combination with an acid buffer (such as citrate buffer or acetate
buffer) enables the detection of acid phosphatase activity.
The ELF-97 substrate is a fluorogenic substrate that has been used for location of
phosphatase activity in (for example) fixed, cultured cells from rat (Telford et al.
1999) and zebrafish (Cox and Singer 1999), marine phytoplankton (González-Gil
et al. 1998), and mycorrhizal fungi (Van Aarle et al. 2001, 2005; Alvarez et al.
2004). The substrate is normally slightly fluorescent in the blue range. However,
once its phosphate is enzymatically removed the substrate forms a crystalline
precipitate, which is bright green fluorescent, indicating the site of activity
(Huang et al. 1993; Larison et al. 1995). Hence the process is called enzymelabelled
fluorescence (ELF). The precipitate has a maximum emission at 530 nm,
and is separated by more than 100 nm from its maximum excitation at 345 nm
(Molecular Probes 2004). This specificity of the ELF substrate makes the signal
clearly distinguishable from the autofluorescence of biological material such
I.M. van Aarle (*)
Universite´ catholique de Louvain, Unité de Microbiologie, Croix du Sud 3/6, 1348, Louvain-la-
Neuve, Belgium
e‐mail: imvaarle@hotmail.com
A. Varma and A.C. Kharkwal (eds.), Symbiotic Fungi, Soil Biology 18, 89
DOI: 10.1007/978‐3‐540‐95894‐9_6, # Springer‐Verlag Berlin Heidelberg 2009