Symbiotic Fungi: Principles and Practice (Soil Biology)

166 D. Krüger et al.
3. From the first dilution after proper mixing, take a further 1 ml and transfer to a
fresh dilution blank, hence getting a 1:100 dilution. Continue the serial dilution
until the desired dilution is obtained.
4. Each suspension is shaken by hand for a few seconds and is drawn into a pipette
and pour-plated or spread-plated (Fig. 10.3). 1 ml of the desired dilution is
transferred to the Petri dish by micropipette or glass pipette. 15–20 ml medium
is added into the Petri plate, cooled just above solidifying temperatures. The
Petri dishes are swirled clockwise and anti-clockwise in order to disperse diluted
soil sample in medium.
5. In case of spread plating for isolating surface-growing fungi 0.1–0.5 ml of the
desired dilution is transferred onto the solidified agar plates. The suspension
is spread over the surface of agar with help of a spreader or sterilized 2–3 mm
diameter glass beads.
6. Incubate at 24–30 C for 6–14 days. The colonies are counted on colony counter
and average number of colonies per dish is multiplied by dilution factor to obtain
the number of microorganisms per gram of the original soil sample (Mukerji
et al. 1998).
Fig. 10.3 Schematic presentation showing pour plate method and direct plate method for isolation
of fungi