Detection, isolation and characterisation of food-borne fungi
R. A. SAMSON, E. S. HOEKSTRA, F. LUND, O. FILTENBORG and J.C. FRISVAD -
Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands and
BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark
For a more detailed description see Introduction to food borne fungi
Direct examination
When fungal growth has been detected by direct examination, the fungus is
streak-inoculated on an appropriate medium. This is preferably done with the aid
of a stereomicroscope.
Direct plating.
This is considered to be the more effective technique for mycological
examination of all foods. For foods such as grains and nuts, a surface
disinfection before direct plating is in most situations, considered essential,
to permit enumeration of fungi actually invading the food.
Surface disinfection: food particles are surface disinfected by vigorous shaking
in 0.4% freshly prepared chlorine for 2 minutes. A minimum of 100 particles should
be disinfected and plated on each chosen medium. The chlorine must only be used
once.
Rinse: after pouring off the chlorine, rinse once in sterile distilled or deionised
water.
Plating: as quickly as possible, transfer food particles with a sterile forceps
to previously poured and set plates, at the rate of 5-10 particles per plate.
Incubation: the standard incubation regime for general-purpose enumerations is 25°C
for 5 days. Plates should be incubated upright. The plates can be kept in perforated
plastic bags to minimise evaporation. The perforation of the plastic bags, combined
with a forced airflow through the incubator is necessary to maintain the initial
composition of the atmosphere in contact with the plates. It has been shown that
accumulation of CO2 significantly influences the growth of fungi.
Results: express results as percentage of particles infected by fungi. Differential
counting of a variety of genera and sometimes even species is possible using a stereomicroscope.
Dilution plating.
Sample sizeDilution plating
Sample size: as large a sample as possible should be used. We normally recommend
5 g samples for homogeneous food materials (like flour) and 40 g samples for not
homogeneous food materials (like grains).ilution should be 1 + 9 in 0.1% peptone.
Soaking: Dried samples, where the fungi are deep seated or internal (e.g. as in
grains and nuts), should be soaked for 30 minutes in 0.1% peptone at room temperature
before stomaching or blending. For powders and other homogeneous samples, no soaking
is required
Homogenisation: use of Stomacher is preferred. If the food material is tough, a
blender may be used. Homogenisation in 2 minutes is recommended if a Stomacher is
used; 1 minute if a blender is used.
Further dilutions: 1:10 (= 1+9) in 0.1% peptone. We recommend 1:5 (= 1+4) in 0.1%
peptone as an alternative, which is especially useful when small concentrations
of fungi are present. Normally a maximum dilution of 10-3 is sufficient, however
if the food has been in contact with soil a maximum dilution of 10-5 may be necessary.
Plating: spread plates are recommended over pour plates. Inocula should be 0.1 ml
per plate.
Incubation: The standard incubation regime for general purpose enumerations is 25°C
for 5 days. Plates should be incubated upright.
2. GENERAL MEDIA
DRBC and DG18.
2 GENERAL MEDIA
DRBC and DG18
Dichloran Rose Bengal Chloramphenicol agar (DRBC; King et al., 1979) and
Dichloran 18% Glycerol agar (DG18; Hocking and Pitt, 1980) are recommended as
general purpose isolation and enumeration media for foods of high water
activity, i.e. aw> 0,90. However the following points should be taken into
consideration:hown
that this medium yields lower counts because basidiomycetous yeasts are often present.
Media containing Rose Bengal are light sensitive. It is therefore important to keep
light exposure to Rose Bengal containing media well below a total of 2 hours during
preparation, storage, inoculation and incubation.
Media should be of approximately neutral pH and contain appropriate antibiotics.
The media are designed for use in dilution plating. By direct plating overgrowth
of fast growing species may occur, especially if surface sterilisation is omitted.
This is due to the addition of considerable amounts of nutrients to the media from
the food product .
Aflatoxigenic species
We recommend the AFPA medium (Pitt et al., 1983), (with both chlortetracycline and
chloramphenicol) for detection of the common aflatoxin producing species as they
are easily differentiated from other species by their bright cadmium orange reverse.
Aflatoxin is not produced on this medium, but rather indicates the production of
a Ferri chelate of aspergillic acids. Sporulation on the medium is rather poor,
but confirmation of identity and aflatoxin production can be done on the media YES
or CYA.
Selection of media for different foods
The choice of media for mycological examination of foods should depend on the associated
mycobiota of the foods or feedstuffs examined. For fruits, vegetables, herbs and
fresh cereals DRBC, DRYES and CZID appear to be preferable (Andersen et al., 1996).
For stored cereals, spices and nuts DG18 at 25°C, DYSG at 20°C and AFPA at 30°C
is an effective combination to be able to count selectively important mycotoxin
producers. In some cases AFPA could be used for fresh (surface disinfected) cereals,
as A. flavus is known to grown and produce aflatoxin on stressed corn plants in
the field in warmer climates. For dairy products, meat and bread CREAD and DG18
is a good combination of media. ADYS is especially suited for preserved foods and
rye bread in combination with DG18.
4. METHODS FOR YEASTS.
General methods for enumeration of yeasts
For products such as4 METHODS FOR YEASTS
General methods for enumeration of yeastsum such as Tryptone Glucose Yeast extract agar (TGY) plus an
antibiotic is recommended. Recommended antibiotics are chloramphenicol and oxytetracycline.
For products where yeasts must be enumerated in the presence of moulds, DRBC is
recommended.
Preservative resistant yeasts
More work is needed to establish the validity of the methods in current use. Media
containing 10% glucose appears to be better than other media. A collaborative study
recommended the following media:
1. TGY + 0.5% acetic acid
2. MEA + 0.5% acetic acid
3. any other medium currently in use.
Diluents for yeasts
For yeasts in high aw foods and beverages, the re-commended diluent is 0.1% peptone.
However, there is no evidence that 0.85-0.9% saline peptone is disadvantageous,
and the use may be continued.
For concentrates, syrups and other low aw samples, a diluent containing at least
20 to 30% glucose is recommended. More work is needed on these and other glucose
concentrations (e.g. 40, 50%) to determine the optimum concentration.
Detection of low numbers of yeasts
Membrane filtration is the recommended method for detection of low numbers of yeasts.
MPN techniques are not recommended. Emerging technologies may eventually produce
better methods.
5. ENUMERATION OF HEAT RESISTANT FUNGI
Recommended method: At least 100 g of product should be heated at 75°C for 30 minutes
and dispersed in an equal volume of double strength agar containing antibiotics.
Malt extract agar (MEA) is recommended. The antibiotics suggested are chlor-amphenicol
(50 ppm) and chlortetracycline (50 ppm). The entire sample should be plated and
incubated, and counted after at least 14 days (to up to 30 days) incubation at 25°C.
In our experience an incubation temperature of 30°C will allow a faster sporulation
of heat resistant Ascomycetes.
Because of the long incubation time and other practical problems of sample preparation,
the following method has been used by
CBS which allows analysis of samples which
are difficult to solve and allows faster detection of potential and common heat
resistant contaminants.
Because of the low concentration of the heat resistant fungi in the sample, it is
still necessary to investigate a large amount of the sample.
For samples, such as pectin, which are difficult to dissolve, aseptically weigh
out 12.5 grams and transfer the sample into a sterile Stomacher bag. Add 250 ml
of Ringer’s solution to the bag and blend the sample. For other samples weigh out
100 grams and transfer the sample into a sterile Stomacher bag. Add 150 ml of Ringer’s
solution to each bag and blend the sample. After blending, seal the Stomacher bag.
1. Heat-treat the Stomacher bags (with the sample) for 30 minutes at 75°C (the sample
should be at 75°C before the 30 minutes when time frame starts). After the heat-treatment,
cool the samples to 50°C.
2. Aseptically transfer the content of the Stomacher bag in a Schott Duran bottle
(500 ml) with 250 ml double strength MEA (50°C). Add the antibiotics.
3. Mix thoroughly.
4. Pour the sample into sterile plastic 14 cm diam Petri-dishes (approx. 8 dishes)
5. Place the Petri-dishes in an upright position in the incubator (28°C, darkness)
6. Examine the Petri-dishes after 7 days and if visible mould structures are present,
isolations on Oatmeal Agar should be made. Re-examine the Petri-dishes after a prolonged
incubation period (up to 14 days and longer).
7. To investigate 100 grams of each sample this procedure should be repeated 7 times.