Enumeration of yeasts and moulds and production of toxins

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Philip B. Mislivec and Michael E. Stack

The large and diverse group of microscopic foodborne yeasts and moulds (fungi) includes several hundred species. The ability of these organisms to attack many foods is due in large part to their relatively versatile, environmental requirements. Although all yeasts and moulds are obligate aerobes (require free oxygen for growth), their acid/alkaline requirement for growth is quite broad, ranging from pH 2 to above pH 9. Their temperature range (10-35°C) is also broad, with a few species capable of growth below or above this range. Moisture requirements of foodborne moulds are relatively low; most species grow at a water activity (aw) of 0.85 or less, although yeasts generally require a higher water activity.

Both yeasts and moulds cause various degrees of deterioration and decomposition of foods. They can invade and grow on virtually any type of food at any time, e.g., they invade field crops such as small grains, nuts, beans, tomatoes, and apples both in the field before harvesting and during storage. They also grow on processed foods and food mixtures. Their detectability in or on foods depends on food type, organisms involved, and degree of invasion, i.e., the contaminated food may be slightly blemished, severely blemished, or completely decomposed, with the actual yeast or mould growth manifested by rot spots of various sizes and colors, unslightly scabs, slime, white cottony mycelium, or highly colored sporulating mould. Abnormal flavors and odors may also be produced. Occasionally a food appears to be mould-free but upon mycological examination, is found to be contaminated. Contamination of foods by yeasts and moulds can also result in substantial economic losses to producer, processor, and consumer.

Several foodborne moulds, and possibly yeasts, may also be a potential hazard to human or animal health because of their ability to produce toxic metabolities known as mycotoxins. Most mycotoxins are stable compounds that are not destroyed during food processing or home cooking. Even though the generating organisms may not survive food preparation, the preformed toxin may still be present. Certain foodborne moulds and yeasts may also be a hazard because of their ability to elicit allergic reactions or even cause infection. Although most foodborne fungi are not infectious, some species can cause infection, especially to vulnerable population groups, e.g., the aged and debilitated and individuals who are receiving chemotherapy or antibiotic treatment.

The dilution plating and the direct plating methods may be used to detect fungi in foods. The dilution plating method, which is the traditional method used in examining foods, is given here. It varies only slightly from the method described in previous editions of the Bacteriological Analytical Manual. The direct plating method is included here because we have found it to be more efficient than the dilution plating method for detecting individual mould species, including most of the toxin producers. (Note: It is less effective in detecting yeasts.) It can also be used to determine whether the presence of mould is due to external contamination or internal invasion. Methodology for testing the ability of isolates of toxigenic mould species to produce mycotoxins on sterile rice water substrate is also included.

Enumeration of Yeasts and Moulds in Foods-Dilution Plating Technique

A. Equipment and materials

1. Basic equipment (and appropriate techniques) for preparation of a food sample homogenate as described in Chapter 2
2. Equipment for plating samples as described in Chapter 4
3. Incubator set at 22-25°C
4. Arnold steam chest
5. pH meter

B. Media and reagents

1. Potato dextrose agar (M114), commercially available in dehydrated form
2. Potato dextrose-salt agar (M114). Same medium as above, amended with 75 9 NaCI. This medium requires 20 9 agar rather than 15 g agar per lifer.
3. Malt extract agar (M78), commercially available in dehydrated form
4. Plate count agar (standard methods) (M112)
5. Tartaric acid solution, 10%, sterile
6. Antibiotic solution (s), see C-2a, below

C. Analysis of samples

1. Prepare sterile agar medium (250 ml portions in prescription bottles or flasks, autoclaved 15 min at 121°C and 15 psi). Temper to 45 ± 1°C in water bath. Prepare medium well in advance and let solidify before remelting and tempering. Do not re-melt solidified medium more than once or under pressure. An Arnold steam chest is recommended. Once medium has been tempered, it can be held for 2-3 h before use, provided water level of water bath is 2-3 cm above surface of agar in aliquot container. Medium of choice is potato dextrose agar, although other media listed above may be used. Potato dextrose-salt agar is especially useful for analyzing samples containing "spreader" moulds (Mucor, Rhizopus, etc.) since the added NaCI effectively inhibits their growth but readily allows detection of other yeast-mould propagules.
2. To inhibit bacterial growth, amend agar medium with either antibiotics or sterile 10% tartaric acid solution (to be done after agar has been tempered and immediately before pouring plates) as follows:

1. Antibiotics. Use of antibiotics is preferred to tartaric acid solution because stock solutions are relatively easy to prepare yeast and mould species, does not result. Chiortetracycline-HCl, at agar medium concentration of 40 ppm, is recommended. Other antibiotics may be used (e.g., chloramphenicol, streptomycin) but should always be used at the same concentration as chlortetracycline-HCl and in addition to it. Prepare stock solutions by dissolving 1 9 of antibiotic in 100 ml of sterile distilled water and filtering through a 0.45 µm membrane (Nalge Sybron Corp., Rochester, NY). Store stock solutions in dark at 4-8°C. Shelf life should exceed 1 month. Equilibrate stock solutions to room temperature immediately before use. If agar medium is in 250 ml aliquots, add 1 ml of 100 ml stock solution to obtain 40 ppm concentration. If medium aliquots are greater or less, adjustments will be necessary.
2. Tartaric acid solution. A 10% solution may be used to adjust agar medium to pH 3 5 ± 0.1 Sterilize solution by filtering through 0.45 µm membrane. Titrate to determine amount of solution needed to adjust pH to 3.5. Type and aliquot volume of medium will affect amount of solution needed. After adding solution to medium, verify pH by letting a portion of medium solidify and checking with pH meter. Do this for every new lot of medium prepared.

3. Prepare food homogenate (Chapter 2) and make appropriate dilutions (Chapter 4). Dilutions of 10(-6) should suffice.
4. Use sterile cotton-plugged pipes to place 1 ml portions of sample dilutions into prelabled 15 x 100 mm petri plates (plastic or glass), and immediately add 20-25 ml tempered agar medium containing either antibiotic (s) or tartaric acid solution. Mix contents by gently swirling plates clockwise then counterclockwise, taking care to avoid spillage on dish lid. Add agar within 1-2 min after adding dilution. Otherwise, dilution may begin to adhere to dish bottom (especially if sample is high in starch content and dishes are plastic) and may not mix uniformly. Plate each dilution in triplicate, using wide bore pipets. From preparation of first sample dilution to pouring of final plate, no more than 20 min. preferably 10 min. should elapse.
5. Incubate plates in dark at 22-25°C. Do not stack plates higher than 3 and do not invert. Let plates remain undisturbed until time for counting.
6. Count plates after 5 days of incubation. Do not count plates after 3 days since handling of plates could result in secondary growth from dislodged spores, making 5 day counts invalid. Count plates containing 10-150 colonies. If mainly yeasts are present, plates with 150 colonies are usually countable. However, if substantial amounts of mould are present, depending on the type of mould, the upper countable limit may have to be lowered at the discretion of the analyst. Report results in colonies (col)/g or (col)/ml based on an average count of the triplicate set. Round off counts to 2 significant figures. If third digit is 6 or above, round off to digit above (e.g., 456 = 460); if 4 or below, round off to digit below (e.g., 454 = 450). If third digit is 5, round off to digit below if first 2 digits are an even number (e.g., 445 = 440); round off to digit above if first 2 digits are an odd number (e.g., 455 = 460).

ENUMERATION OF MOULDS IN FOODS-DIRECT PLATING TECHNIQUE FOR FOODS (SUCH AS DRIED BEANS, NUTS, WHOLE SPICES, COFFEE AND COCOA BEANS) THAT CAN BE HANDLED WITH FORCEPS

A. Equipment and materials

1. Freezer, -20°C
2. Beakers, sterile, 150 ml
3. Forceps, sterile
4. Arnold steam chest
5. Water bath, 45 ± 1°C
6. Incubator, 22-25°C

B. Media and reagents

1. Potato dextrose-salt agar (M114)
2. Antibiotic solution
3. NaOCI solution, 5%
4. Sterile distilled water

C. Analysis of non-surface-disinfected (NSD) foods

1. Before plating. Hold samples at -20°C for 72 h to kill mites and other insects that might interfere with analysis.
2. Preparation of agar plates. Use potato dextrosesalt agar (containing 75 9 Nacl/liter). NaCl inhibits growth of mould "spreaders" and prevents germination of viable seeds which otherwise could cause petri dish lid and stack disorientation. To tempered agar, add 40 ppm chlortetracycline-HCI (C-2a, above). Into 15 x 100 mm petri plates (plastic or glass) pour about 30 ml medium and let solidify. Because of prolonged incubation time, more medium is needed for direct plating than for dilution plating in each petri dish. Prepare 10 plates for each sample to be analyzed. Plates may be prepared in advance, but period between preparation and use should not exceed 24 h.
3. Plating of sample. From each sample, transfer about 50 9 into sterile 150 ml beaker. Using 95% ethanol-flamed forceps, place intact food items on surface of solidified agar, 5 items per plate (1 in plate center and 1 in each quadrant), 50 items total per sample. Flame forceps between plating of each item. Use several forceps alternately to avoid overheating. Do not plate visibly mouldy or otherwise blemished items.
4. Incubation of plates. Align plates in stacks of 10; identify top and bottom plate of each stack with sample number plus date of plating. Incubate stacks, undisturbed, in dark at 22-25°C for 1421 days.
5. Reading of plates. Determine occurrence of mould in percentages (e.g., if mould emerged from all 50 food items, mouldiness is 100%, if from 32 items, mouldiness is 64%). Determine percent occurrence of individual mould genera and species in like manner. Several Asperigillus species (or species complexes) plus most other foodborne mould genera may be identified directly on above medium by experienced analysts with low power (10-30X) magnification.

D. Analysis of surface-disinfected (SD) foods

Perform disinfection in clean laboratory sink, not stainless steel, free from any acid residues, with tap water running (precautions against chlorine gas generation). Using rubber gloves, transfer about 50 9 of sample into sterile 150 ml beaker. Cover with 5% NaOCI solution for 1 min. swirling beaker contents gently but constantly in clockwisecounterclockwise motion Decant 5% NaOCI solution and give beaker contents three 1 min sterile distilled water rinses. Prepare plates, plate sample, incubate, and read plates as in C, 2-5, above. Comparison of NSD and SD results from same sample will indicate whether mouldiness was due mainly to surface contamination or to internal invasion and growth.

METHODS FOR DETERMINING TOXIN PRODUCTION BY MOULDS

A. Equipment and materials

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 (S & S No. 588)
5. Boiling chips, silicon carbide
6. Fume hood equipped with steam bath; airflow 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

B. Media and reagents

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. NaOCI solution, 5%

C. Toxin production

Into 300 ml wide-mouth Erlenmeyer flask, add 50 9 rice and 50 ml distilled water. Cotton plug flasks and autoclave 20 min at 121°C and 15 psi. Aseptically multispore-inoculate separate cooled flasks with individual mould isolates. Incubate inoculated flasks at 22-25°C until entire surface is covered with growth and mycelium has penetrated to bottom of flask (15-20 days). To each flask, add 150 ml chloroform (150 ml methanol if toxin in question is deoxynivalenol), using short-stem glass funnel inserted alongside unremoved cotton plug (to minimize mould spore dissemination). Hear flask contents in fume hood on steam bath until solvent begins to boil. (Conduct all subsequent steps in fume hood.) With spatula, break up mouldy rice cake and transfer flask contents into explosionproof blender and blend at high speed for 1 min. Filter blender contents through filter paper inserted into short-stem glass funnel. Collect filtrate in 300 ml Erlenmeyer flask. Return rice cakes to blender, add 100 ml unheated solvent and blend 1 min at high speed. Filter as above and combine filtrates. Add boiling chips to flask containing filtrates and steam evaporate to 20-25 ml. If analysis is not to follow immediately, evaporate to dryness and store flask in dark. Rinse all glassware, etc., used for extraction in 5% NaOCI solution before soap and water cleansing. Rice cake should be submerged in 5% NaOCI solution for 72 h before autoclaving and disposal.

D. Toxin analysis

Toxin analysis requires use of appropriate mycotoxin standards for both qualitative and quantitative evaluation. Use either thin layer chromatography or high performance liquid chromatography, as described in Chapter 26, of Official Methods of Analysis 14th ea., 1984 (Association of Official Analytical Chemists, Arlington, VA), for determination of most foodborne mycotoxins.

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