The next day, the agar plate surface is checked to see whether spores have
been ejected or dropped. If spores are present on the agar surface, their identity
can be established by preparing a slide of agar containing spores. If the spore is
the correct one, a sterilized glass needle can be used to transfer a piece of agar
containing each spore onto the PDA plate as before.
The PDA plate is incubated in 25 C until their colony diam. are about 1 to
2 cm. A small piece of mycelium with agar is then cut and transferred t6
another PDA plate and the culture is checked after few days, if there is no
contamination, a pure culture has been obtained.
If there is any contamination in a culture, subcultures may be obtained by
transferring small threads of uncontaminated hyphae with the glass needle.
Although difficult, it is possible to rescue cultures by this method.
Storage of cultures
Once a pure culture has been obtained, it can be maintained on an agar
slant at 4 C or stored as a few pieces of agar with mycelia in cryo vials with
10% glycerol in liquid nitrogen. Alternatively, it can be stored as a few pieces
of agar with mycelia in small bottle with sterilized parafin oil or sterilized
water at 4 C. There are several other methods to maintain fungal cultures and
these can be reviewed in the "Preservation and Maintenance of Living Fungi"
(Smith and Onions, 1994). It is recommended that a mixture of methods are
used so that preservation of a culture is more likely (in case one method fails).