Symbiotic Fungi: Principles and Practice (Soil Biology)

11 Isolation of Metabolically Active Arbuscules and Intraradical Hyphae 193
11.2.3.3 Protocol
The isolated arbuscule and hyphal fraction are subjected to the assessment based on
histochemical observation of SDH, alkaline phosphatase (ALP) and acid phosphatase
(ACP) according to the methods described elsewhere (McDonald and Lewis
1978; Saito et al. 1993; Saito 1995).
1. Incubate arbuscules and hyphae at 35 C for 2 h in the following solutions: SDH;
0.25 M sodium succinate, 0.05 M Tris–HCl buffer (pH 7.6), 0.5 mM MgCl2, 1 mg ml 1 Nitroblue tetrazolium; ALP; 4 mM a-naphtyl acid phosphate,
0.6 mg ml 1 Fast Blue RR, 0.1 M Tris–HCl buffer (pH 8.5); ACP; as ALP
but using the buffer 0.1 M sodium acetate.
2. Wash arbuscules with deionized water after incubation of the arbuscules, treat
the arbuscule fractions for SDH, ALP and ACP staining by counter staining with
0.5 mg ml 1 acid fuchsin, transfer to lactoglycerol and mount on glass slides.
3. Count the number of arbuscules and hyphae under the light microscope. Then
count the number of arbuscules with SDH, ALP or ACP activity, and those
without activity separately.
4. To evaluate the loss in the SDH, ACP and ALP activity of arbuscule during the
isolation process, the percentage of SDH, ACP and ALP active arbuscules in the
roots can be compared.
5. Squash the stained root pieces on a slide glass and measure the percentage of the
arbuscules showing SDH, ACP or ALP activity.
Fig. 11.1 Arbuscules isolated from root of a mutant har1 colonized by Glomus sp. and stained
for succinate dehydrogenase (SDH) activity. SDH activity of arbuscules indicated with arrows.
Bar indicates 50 mm