Advances in Food Mycology

Standardization of Methods for Detecting Heat Resistant Fungi 109
mended. At least 100 g of sample should be examined (Samson et al.,
2000).
Homogenize the sample before beginning the analysis. Transfer 100
g of sample into a sterile Stomacher bag. Add sterile water (150 g) to
the sample and homogenize using a Stomacher for 2 to 4 min. If the
sample is likely to contain a higher concentration of heat resistant
fungi (e.g. a soil sample), or the sample cannot be homogenized in 150
grams of water (e.g. solid ingredients such as pectin), then a smaller
amount of sample can be used. After the homogenization step, the
Stomacher bag should be sealed about half way along its length using
a heat-sealer, ensuring that no air bubbles are present. After checking
the bag for leakage, heat treat the Stomacher bag for 30 min at 75°C
in a water bath (preferably one with the capability of shaking or stirring).
The water-bath should be at 75°C before the sample is introduced.
The sample should be placed in a horizontal position, totally
submerged in the water.
After the heat treatment, cool the samples to approximately 55°C.
Aseptically transfer the contents of the Stomacher bag to a Schott
bottle, or similar, (500 ml) with 250 ml melted double strength MEA
containing chloramphenicol (200 mg/l, Oxoid) tempered to 55°C. Mix
thoroughly and distribute the agar and sample mixture into seven
large plastic Petri dishes (diameter 14.5 cm). Place the Petri dishes into
a polyethylene bag to prevent drying and incubate in an upright
position at 30°C in darkness.
The general procedure is illustrated in Figure 1.
2.3. Incubation
Many protocols require plates to be incubated for at least 30 days.
This period is too long for quality control in the food and beverage
industry. In our experience, heat-resistant fungi will usually form
colonies after 5 days and mature within 14 days incubation at 30°C, so
check the Petri dishes for the presence of colonies after 7 and 14 days.
Subculture if necessary, and identify all colonies using standard methods
(Pitt and Hocking, 1997; Samson et al., 2000). In some cases a
prolonged incubation period is necessary for the identification of
the fungus. An overview of the incubation time needed for some
particular species is given in Figure 2.