Methods

GENERAL PURPOSE METHODS

Direct examination

In foods spoiled by fungi it is often possible to ob-serve the responsible fungal growth directly by the naked eye followed by observations in a stereomi-croscope. This is due to the size of the fungal col-ony and that fungal growth normally takes place at the surface of the product. However the observation has to be made immediately after growth has taken place, since any handling of the product is bound to remove the visual appearance of the fungi. If fungal growth can be expected, it is always recommended to examine the food products by microscopy. Slides can be prepared by putting a small part or volume in a mounting medium (e.g. lactic acid with aniline blue). Preparations with the aid of adhesive tape as described on page 2 can also be helpful.

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 tech-nique for mycological examination of all foods. For foods such as grains and nuts, a surface disinfec-tion before direct plating is in most situations, con-sidered essential, to permit enumeration of fungi actually invading the food. An exception is to be made for cases where surface contaminants be-come part of the downstream mycobiota, e.g. wheat grains to be used in flour manufacture. In such cases grains should be investigated both with and without surface disinfection.

Surface disinfection: food particles are surface dis-infected by vigorous shaking in 0.4% freshly pre-pared 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 parti-cles 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 gen-eral-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 incuba-tor 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 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).

Initial dilution: the initial dilution 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 blend-ing. 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 dilu-tion 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.


GENERAL MEDIA (for formulations).

DRBC and DG18

Dichloran Rose Bengal Chloramphenicol agar (DRBC; King et al., 1979) and Dichloran 18% Glyc-erol agar (DG18; Hocking and Pitt, 1980) are rec-ommended as general purpose isolation and enu-meration media for foods of high water activity, i.e. aw> 0,90. However the following points should be taken into consideration:
1. DG18 is less suitable for fresh fruits and vege-tables, where studies have shown that this me-dium yields lower counts because basidiomyce-tous yeasts are often present.
2. Media containing Rose Bengal are light sensi-tive. Inhibitory compounds are produced in sig-nificant concentrations after 2 hours exposure to light (P.V.Nielsen, personal communication). It is therefore important to keep light exposure to Rose Bengal containing media well below a total of 2 hours during preparation, storage, in-oculation and incubation.
3. Media should be of approximately neutral pH and contain appropriate antibiotics.
4. We recommend a mixture of 2 antibiotics to be used instead of higher concentrations of a sin-gle compound. In our experience e.g. 100 ppm chloramphenicol is often insufficient to inhibit growth of bacteria from food products like vege-tables, spices, cereals and meats. We recom-mend the combined use of chloramphenicol (50 ppm) and chlortetracycline (50 ppm). Chloram-phenicol is heat-stable and may be added be-fore autoclaving. Chlortetracycline is heat labile and must be added to the media after autoclav-ing as a filter sterilised solution. It is relatively unstable in solution and thus must be freshly prepared or refrigerated. Gentamycin is not recommended, as it has been reported to cause inhibition of some yeast species.
5. The media are designed for use in dilution plat-ing. By direct plating overgrowth of fast growing species may occur, especially if surface sterili-sation is omitted. This is due to the addition of considerable amounts of nutrients to the media from the food product .


SELECTIVE MEDIA

It is important to notice that all isolation media are selective to some degree, simply because of differ-ent competition abilities of the different species when they grow on the agar medium itself. Plant pathologists have solved that problem by using blotter tests and could, because of the rather few well known plant pathogenic species in anyone genus, often identify fungi to species level (Benoit and Mathur, 1970; Nath et al., 1970; Chidambaram et al., 1971; Kulshrestha et al., 1976; Agarwall et al., 1989) This is not possible for food or air-borne fungi as there are many species that cannot be differentiated or identified directly on seeds or other food items, especially the Penicillia and Fusaria. When using combinations of extract from cereals, fruits or vegetables with (enzymatically digested) protein sources and carbohydrates or chemically well defined media combined with different tem-peratures, some selection is thus unavoidable Two major principles for the development of selective media is inhibition of bacterial growth and reducing colony diameters of fast growing fungi (King et al., 1986; Beuchat, 1992a). Further selective principles may be introduced to select for more specific groups of fungi or even particular fungal species. These are dealt with below. The efficiency of media may also depend on the isolation method em-ployed. Stronger selective principles are needed for direct plating than for dilution plating.