The most common problems when isolating fungi are contamination by
bacteria and yeasts, and the fact that the wrong species may inadvertently be
isolated. To overcome these problems, dilution of spore masses can be used to
reduce the chances of yeast contamination and different antibiotics (e.g.
penicillin, 0.5 g/L, streptomycin, 0.5 g/L) can be used to reduce bacterial
contamination. There are several methods to ensure that the correct single spore
is isolated (Hansen, 1926) and a commonly used method is reported here.
Unfortunately, not all spores will germinate in artificial media, and other
techniques may be required for successful isolation of these fungi. Fungal
cultures stored in a high nutrient medium, such as Potato Dextrose Agar (PDA)
may lose their ability to produce enzymes or metabolites, or perform other
functions. Care is therefore needed in selecting a storage medium (Smith and
Onions, 1994).
General material
For effective isolations to be carried out, an isolation room or clean bench
with as little air movement as possible is desirable. A dissection microscope
with illumination from both below and above, kept permanently in a lamina
flow cabinet is desirable. A compound microscope can be kept near by to check
spores and to observe germinating spores). This will reduce the likelihood of
contamination. Alcohol (70%) is used to clean the working surface and some
sort of burner is required for sterilizing utensils.
In the methods recommended below, water agar (WA) and PDA are
routinely used as inexpensive media. Other media may be required for difficult
fungi, such as V8 juice agar for lower fungi, or corn meal agar (CMA) when
sporulation is required in lignicolous fungi. Sterilised water and sterilised
Pasteur pipettes must also be prepared beforehand.