Soil Microbial Ecology - Soil Molecular Ecology Laboratory

Page 68
mechanical shaker. Prepare dilutions as in exercise #1 and Plate in bacteria (start at 10 -3
because less soil goes into the first dilution compared with soil dilution lab).
To determine weight of rhizosphere soil, the roots are removed from the original dilution
flask, washed, and the wash water is collected in the original flask. The water is
evaporated and the soil residue is dried to constant weight in an oven at 105-110 o C. The
flask containing dry soil is weighed and dilution factors are calculated, allowance being
made for the amount of soil removed in preparing the dilutions.
Count bacterial colonies at appropriate time intervals during the week and in the
following laboratory session. Describe the morphology of typical colonies. How do
these counts compare to the bulk soil counts in Exercise #1?
2. DIRECT MICROSCOPY.
Obtain 5 roots (< 2 mm diam.) and gently wash to remove excess soil. Place washed
roots in FITC solution (see page 27) for several minutes. Wash roots in sodium
carbonate buffer (5 changes). Cut roots into 1-2 cm lengths and mount in glycerol
adjusted to pH 9.6 with buffer. Observe roots with epifluorescent microscopy (2170
McC).
REFERENCE:
Chapter 2 in Metting's Soil Microbial Ecology