Symbiotic Fungi: Principles and Practice (Soil Biology)

6 Assessment of Phosphatase Activity Associated with Mycorrhizal Fungi 91
l Slides and cover glasses
l Fine forceps
l Nail varnish
6.3.3 Solutions
A couple of solutions are needed before starting an ELF-assay. Some can be
purchased, whereas others should be prepared in advance.
In order to visualise alkaline phosphatase an alkaline buffer is needed. Most
appropriate is to use the alkaline detection buffer from the ELF kit. However, if
another alkaline buffer is used (such as, e.g., Tris-HCl) one should pay attention
that the precipitate dissolves at a pH above 8.0–8.5. In order to visualise acid
phosphatase a buffer with a low pH should be used. It is possible to use a
commercial citrate buffer, such as the one from Sigma with pH 4.8 (90 mM).
Otherwise an acetate or citrate buffer can be used and in that case the pH can be set
at a specific value. These buffers are prepared as follows:
1. Acetate buffer, pH 5.5, 0.2M:
l 0.2M acetic acid solution by dissolution of 12.10 g acetic acid in dH 2O, fill
up to 1 l
l 0.2M sodium acetate solution by dissolution of 16.46 g sodium acetate in
dH2O, fill up to 1 l
l Mix 0.2M acetic acid solution into 0.2M sodium acetate solution until pH 5.5
is reached
2. Citrate buffer, pH 4.0–6.0, 0.1M:
l 0.1M solution of citric acid by dissolution of 21.01 g citric acid in dH 2O, fill
up to 1 l
l 0.1M solution of sodium citrate by dissolution of 29.41 g sodium citrate
(C6H5O7Na3 2H2O) in dH2O, fill up to 1 l
l Mix x ml of citric acid solution with y mL of sodium citrate solution
according to Table 6.1
l Confirm pH
After the samples have been incubated in the ELF substrate-buffer solution and
before mounting the samples on the slide, it is recommended to wash the samples in
Table 6.1 The proportion by which the two components (x = 0.1 M
citric acid solution, y = 0.1 M sodium citrate solution) of the citrate
buffer should be mixed to obtain a pH between 4.0 and 6.0
pH x (ml) y (ml)
4.0 33.0 17.0
4.4 28.0 22.0
4.8 23.0 27.0
5.2 18.0 32.0
5.6 13.7 36.3
6.0 9.5 41.5