Enumeration of Yeasts and Molds in Food

--Dilution Plating Technique

Equipment and Materials

A.

1. Basic equipment (and appropriate techniques) for preparation of sample homogenate, see Chapter 1
2. Equipment for plating samples, see Chapter 3
3. Incubator, 25°C
4. Arnold steam chest
5. pH meter
6. Water bath, 45 ± 1° C

Media and Reagents

B.

Media

1. Dichloran rose bengal chloramphenicol (DRBC) agar (M183)
2. Dichloran 18% glycerol (DG18) agar (M184)
3. Plate count agar (PCA), standard methods (M124); add 100 mg chloramphenicol/liter when this medium is used for yeast and mold enumeration. This medium is not efficient when "spreader" molds are present.
4. Malt agar (MA)(M185)
5. Malt extract agar (Yeasts and Molds) (MEAYM) (M182)
6. Potato dextrose agar (PDA), dehydrated; commercially available (M127)

Antibiotic Solutions

Antibiotics are added to mycological media to inhibit bacterial growth. Chloramphenicol is the antibiotic of choice, because it is stable under autoclave conditions. Therefore, media preparation is easier and faster due to the elimination of the filtration step. The recommended concentration of this antibiotic is 100 mg/liter medium. If bacterial overgrowth is apparent, prepare media by adding 50 mg/liter chloramphenicol before autoclaving and 50 mg/liter filter-sterilized chlortetracycline when the media have been tempered, right before pouring plates.

Prepare Stock Solution

Prepare stock solution by dissolving 0.1 g chloramphenicol in 40 ml distilled water; add this solution to 960 ml medium mixture before autoclaving.

• When both chloramphenicol and chlortetracycline are used, add 20 ml of the above chloramphenicol stock solution to 970 ml medium before autoclaving.

• Then, prepare chlortetracycline stock solution by dissolving 0.5 g antibiotic in 100 ml distilled water and filter sterilize.

Use 10 ml of this solution for each 990 ml of autoclaved and tempered medium.

Refrigerate in the dark and re-use remaining stock solutions for up to a month.

Stock solutions should be brought to room temperature before adding to tempered medium.

Enumeration of Molds in Foods

--Direct Plating Technique for Foods That Can Be Handled with Forceps (Dried Beans, Nuts, Whole Spices, Coffee and Cocoa Beans, etc.)

Equipment and Materials

A.

1. Freezer, -20° C
2. Beakers, sterile, 300 ml
3. Forceps, sterile
4. Arnold steam chest
5. Water bath, 45 ± 1° C
6. Incubator, 25° C

Media and Reagents

B.

1. Dichloran rose bengal chloramphenicol (DRBC) agar (M183)
2. Dichloran 18% glycerol (DG18) agar (M184)
3. Antibiotic solutions (see previous section)
4. NaOCl (commercial bleach) solution, 10%
5. Sterile distilled water

Methods for Determining Toxin Production by Molds

Equipment and Materials

A.

1. Erlenmeyer flasks, 300 ml, wide-mouth
2. Cotton, nonabsorbent
3. Funnels, short-stem glass, 90-100 mm diameter
4. Filter paper, 18 cm diameter, folded (Schleicher & Schuell No. 588)
5. Boiling chips, silicon carbide
6. Fume hood equipped with steam bath; air-flow rate, 100 cubic ft/min
7. Blender, high speed, explosion-proof
8. Thin layer chromatographic apparatus or high-performance liquid chromatograph
9. Incubator, 22-25°C

Media and Reagents

B.

1. Long or short grain polished rice
2. Chloroform for extraction of aflatoxins, ochratoxins, sterigmatocystin, xanthomegnin, luteoskyrin, patulin, penicillic acid, citrinin, T-2 toxin, zearalenone
3. Methanol for extraction of deoxynivalenol
4. Appropriate mycotoxin standards
5. NaOCl solution, 5%