The stock cultures for deep freezing was prepared by using
a basal sawdust medium. The medium contained 1.5 g beech
(Fagus crenata Blume.) sawdust, 0.45 g rice bran, and about
3.5 ml distilled water, and the water content was adjusted to
64%. For the preparation of test media, different concentrations
of cryoprotectants were added to the basal medium.
The media were poured into test tubes (Pyrex), 15.5mm in
diameter and 155 mm in length, pressed to about 5cm in
height, and corked with foamed silicon rubber plugs
(Shirikosen; Shinetsu Chemical, Tokyo, Japan). The cultures
were autoclaved at 120°C for 10 min. To prepare the
inoculum of the mycelium agar block, 16ml agar plate
medium, which consisted of 20 g glucose, hot water extract
of 200 g diced potatoes, 15 g agar, and 1 l distilled water, pH
5.6, was prepared. After autoclaving at 115°C for 5 min, it
was inoculated with the mycelium of the stock culture. The
plate culture was incubated at 25°C in the dark for 7 days.
The mycelial colony of the plate culture was cut by 3 3
3 mm for the inoculum agar blocks, and an agar block was
placed onto each of the test sawdust media. The cultures
were incubated at 25°C to allow mycelial growth for 6 days
in the dark, and they were then moved into a culture room
at 25°C for 3–4 weeks until the mycelia grew over the
media. The cultures were put directly in a deep-freezer set
at 85°C without any program of linear precooling and
stored for the designated duration. The frozen cultures
were immediately thawed for 3h at room temperature when
their viabilities were determined.