BAM Chapter 18 - Yeasts, Molds and Mycotoxins - FDA
BAM Chapter 18 - Yeasts, Molds and Mycotoxins - FDA
April 2001
The large and diverse group of microscopic foodborne yeasts and molds (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 the majority of yeasts
and molds 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 molds are relatively low; most species can grow at a
water activity (aw) of 0.85 or less, although yeasts generally require a higher water activity.
Both yeasts and molds cause various degrees of deterioration and decomposition of foods.
They can invade and grow on virtually any type of food at any time; they invade crops such as
grains, nuts, beans, and fruits in fields 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; the contaminated food may be slightly
blemished, severely blemished, or completely decomposed, with the actual growth
manifested by rot spots of various sizes and colors, unsightly scabs, slime, white cottony
mycelium, or highly colored sporulating mold. Abnormal flavors and odors may also be
produced. Occasionally, a food appears mold-free but is found upon mycological examination
to be contaminated. Contamination of foods by yeasts and molds can result in substantial
economic losses to producer, processor, and consumer.
Several foodborne molds, and possibly yeasts, may also be hazardous to human or animal
health because of their ability to produce toxic metabolites 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 molds and yeasts may also elicit
allergic reactions or may cause infections. Although most foodborne fungi are not infectious,
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some species can cause infection, especially in immunocompromised populations, such as the
aged and debilitated, HIV-infected individuals, and persons receiving chemotherapy or
antibiotic treatment.
The dilution plating and the direct plating methods may be used to detect fungi in foods. The
direct plating method is more efficient than the dilution plating method for detecting
individual mold species, including most of the toxin producers, but it is less effective in
detecting yeasts. It is also used to determine whether the presence of mold is due to external
contamination or internal invasion. Methodology for testing the ability of isolates of toxigenic
mold species to produce mycotoxins on sterile rice water substrate is included here.
3. Incubator, 25°C
5. pH meter
6. Water bath, 45 ± 1° C
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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.
C. Procedures:
Sample preparation
Analyze 25-50 g from each subsample; generally, larger sample sizes increase
reproducibility and lower variance compared with small samples. Test individual
subsamples or composite according to respective Compliance Program for the food
under analysis. Add appropriate amount of 0.1% peptone water to the weighed sample
to achieve 10-1 dilution, then homogenize in a stomacher for 2 min. Alternatively,
blending for 30-60 sec can be used but is less effective. Make appropriate 1:10 (1+9)
dilutions in 0.1% peptone water. Dilutions of 10-6 should suffice.
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Incubate plates in the dark at 25°C. Do not stack plates higher than 3 and do not invert.
Note: Let plates remain undisturbed until counting.
Counting of plates
Count plates after 5 days of incubation. If there is no growth at 5 days, re-incubate for
another 48 h. Do not count colonies before the end of the incubation period because
handling of plates could result in secondary growth from dislodged spores, making final
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 mold
are present, depending on the type of mold, the upper countable limit may have to be
lowered at the discretion of the analyst. Report results in colony forming units (CFU)/g
or CFU/ml based on average count of triplicate set. Round off counts to two 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). When plates from all dilutions have
no colonies, report mold and yeast counts (MYC) as less than 1 times the lowest dilution
used.
Isolate individual colonies on PDA or MA, if further analysis and species identification
is necessary.
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1. Freezer, -20° C
3. Forceps, sterile
5. Water bath, 45 ± 1° C
6. Incubator, 25° C
Before plating, hold sample at -20° C for 72 h to kill mites and insects that might
interfere with analysis.
Prepare DRBC agar as described in the appendix. If DRBC is not available, or the water
activity of the analyzed sample is less than 0.95, use DG18 agar. Media should be
prepared no more than 24 h prior to use.
From each sample, transfer about 50 g into a sterile 300 ml beaker. Using 95% ethanol-
flamed forceps place intact food items on surface of solidified agar, 5-10 items per plate
(depending on size of food item) 50 items total per sample.
Flame forceps between plating of each item. Use several forceps alternately to avoid
overheating. Do not plate visibly moldy or otherwise blemished items.
Align 3-5 plates in stacks and identify with sample number plus date of plating.
Incubate stacks, undisturbed in the dark at 25°C for 5 days. If there is no growth at 5
days of incubation, re-incubate for another 48 h to allow heat- or chemically-stressed
cells and spores enough time to grow.
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Reading of plates
Determine occurrence of mold in percentages. If mold emerged from all 50 food items,
moldiness is 100%; if from 32 items, moldiness is 64%. Determine percent occurrence
of individual mold genera and species in like manner. Experienced analysts may
identify Aspergillus, Penicillium and most other foodborne mold genera directly on
medium with low power (10-30X) magnification.
Methods for counting viable yeasts by plating are described above. A direct microscopic
procedure for counting nonviable and viable yeasts in beverages and other liquid samples is
presented here. Quantitating yeast cells by microscopy eliminates the need for extended
incubation, thus reducing the analytical time required. All yeasts can be counted, and living
and dead yeast cells can be differentiated.
C. Syringes, disposable
D. Pipets
E. Forceps
F. Bibulous paper
H. Fluorescence microscope: blue excitation; l0X eyepieces with Howard mold count or
other eyepiece grid; 20× or 40× objective
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Reagents
Sample preparation for filterable liquids (e.g. water and grape juice)
Filter aliquot (usually 10 ml) of sample through Millipore filter (AABG, 0.8 µm, black,
gridded).(Portion size can be increased or decreased, depending on level of
contamination). Use Millipore disk filter holder which attaches to standard syringe.
Make sure that syringe is accurate. If not, remove plunger, attach syringe to filter
holder, and pipette 10 ml into syringe. Press all of sample through filter. Do this with
air cushion of about 3 ml between plunger and sample. Keep filter holder vertical to
ensure even distribution of sample over filter. Remove filter from filter holder and place
on microscope slide; grid should be parallel to edges of slide to facilitate counting.
Sample preparation for non-filterable liquids that clog the filter (e.g.
orange juice)
To suppress background interference in fluorescence microscope, mix 4 ml sample with
1 ml sodium hydroxide (25 g in 100 ml water). Shake well and wait 10 min. Place
Millipore filter (AABG, 0.8 µm, black, gridded) on a piece of bibulous paper and spread
0.1 or 0.01 ml (depending on level of contamination) of sample over filter. When filter
surface is dry, place filter on microscope slide, keeping grid parallel to edges of slide to
facilitate counting.
Cover filter with a drop of aniline blue, 1% in M/15 (11.6 g/L) K2HPO4, adjusted to pH
8.9 with K3PO4. Spread aniline blue stain over whole filter with glass rod or coverslip
without touching filter itself. Wait about 5 min; then cover filter with 24 × 24 mm
coverslip.
Count yeasts, using fluorescence microscope with blue excitation. Use 10X eyepiece
with Howard mold count or other eyepiece grid, and 20X (or 40X) objective. Count 3
squares of eyepiece grid in each field of filter not covered by gasket. Count budding
yeasts as 1 cell if daughter cell is obviously smaller than mother cell. If they are
approximately equal in size, count them as 2 cells. Count all yeasts located completely
within an eyepiece square and all yeasts touching left and lower border of eyepiece
square. Do not count yeasts touching right and upper borders.
This method also differentiates dead (heat- or formaldehyde-killed) and living yeast
cells. Dead cells show fairly uniform fluorescence, and plasma may be granular. In
living cells, the cell wall stains brighter and is more defined than the plasma, which is
less prominent and uniformly stained.
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Determine area of filter covered by 1 square of eyepiece grid, using objective (stage)
micrometer. For filtered samples, the working area of the Millipore filter (portion not
covered by the gasket) is 380 mm2. For nonfiltered samples, it is the entire filter, or 491
mm2, since no gasket is used.
NOTE: For non-filterable liquids, volume includes only net amount used and not
volume of NaOH added (i.e., 80% of total volume applied to filter).
For background information on the method, including photographs of dead and living
yeast cells, see Koch et al., ref. 8, below.
2. Cotton, nonabsorbent
6. Fume hood equipped with steam bath; air-flow rate, 100 cubic ft/min
9. Incubator, 22-25°C
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5. NaOCl solution, 5%
C. Toxin production
Into 300 ml wide-mouth Erlenmeyer flask, add 50 g rice and 50 ml distilled water. Plug
flasks with cotton and autoclave 20 min at 121°C and 15 psi. Aseptically multispore-
inoculate separate cooled flasks with individual mold 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 mold spore dissemination).
Heat flask contents in fume hood on steam bath until solvent begins to boil. (Conduct
all subsequent steps in fume hood.) With spatula, break up moldy rice cake and transfer
flask contents into explosion-proof 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 evaporate with steam to 20-25 ml. If
analysis is not to follow immediately, evaporate to dryness and store flask in the dark.
Rinse all glassware, etc., used for extraction in 5% NaOCl solution before soap and
water cleansing. Submerge rice cake in 5% NaOCl solution for 72 h before autoclaving
and disposal.
D. Toxin analysis
Appropriate mycotoxin standards are required for both qualitative and quantitative
analysis of toxin. Use either thin layer chromatography as described in references 16 or
17 or high performance liquid chromatography,as described in reference 15a, to
determine mycotoxins extracted from mold cultures. Naturally occurring mycotoxins in
foods or feeds can best be determined by methods described in Official Methods of
Analysis (16).
1. Association of Official Analytical Chemists, 1990. Official Method of Analysis, 15th ed.,
AOAC Arlington, VA.
2. Barnett, H.L. 1960. Illustrated Genera of Imperfect Fungi, 2nd ed. Burgess,
Minneapolis.
3. Beneke, E.S., and A.L. Rogers. 1971. Medical Mycology Manual, 3rd ed. Burgess,
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Minneapolis.
4. Cole, R.J. (ed.) 1986. Modern Methods in the Analysis and Structural Elucidation of
Mycotoxins. Academic Press, Orlando, FL.
6. Gilman, J.C. 1957. A Manual of Soil Fungi, 2nd ed. Iowa State University Press, Ames,
IA.
7. King, A.D. Jr, J.I. Pitt, L.R. Beuchat, and J.E.L. Corry (eds.) 1986. Methods for the
Mycological Examination of Food. Plenum Press, New York.
8. Koch, H.A., R. Bandler, and R.R. Gibson, 1986. Fluorescence microscopy procedure for
quantitation of yeasts in beverages. Appl. Environ. Microbiol., 52: 599-601.
9. Lodder, J. 1970. The Yeasts, a Taxonomic Study, 2nd ed. North-Holland Publishing Co.,
Amsterdam, The Netherlands.
10. Milivec, P.B. 1977. The genus Penicillium, pp. 41-57. In: Mycotoxic Fungi, Mycotoxins,
and Mycotoxicoses, Vol. 1. T.D. Wyllie and L.G. Morehouse (eds.). Marcel Dekker, New
York.
11. Pitt, J.I., A.D. Hocking, R.A. Samson and A.D. King, 1992. Recommended methods for
mycological examination of foods, pp. 365-368. In: Modern Methods in Food Mycology,
R.A. Samson, A.D. Hocking, J.I. Pitt, and A.D. King (eds.). Elsevier, Amsterdam.
12. Raper, K.B., and D.I. Fennell. 1965. The genus Aspergillus. William & Wilkins,
Baltimore.
13. Raper, K.B., and C. Thom. 1968. A Manual of the Penicillia. Hafner, New York.
14. Rodricks, J.V., C.W. Hesseltine, and M.A. Mehlman,(eds.) 1977. Mycotoxins in Human
and Animal Health. Pathotox, Park Forest South, IL.
15. Samson, R.A., A.D. Hocking, J.I. Pitt and A.D. King, 1992. Modern Methods in Food
Mycology. Elsevier, Amsterdam.
16. Scott, P.M. 1995. Chapter 49, Natural Toxins. pp 49-1 to 49-49. Official Methods of
Analysis, 16th ed. AOAC International, Gaithersburg, MD.
17. Stack, M.E. 1996. Toxins, pp. 1033-1045. In: Handbook of Thin-Layer
Chromatography, 2nd ed., J. Sherma and B. Fried (eds.). Marcel Dekker, New York.
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Mix above items and steam to dissolve agar, then bring volume to 1000 ml with distilled
water. Add 220 g glycerol (analytical reagent grade), and sterilize by autoclaving at 121°C for
15 min. Temper medium to 45° C and pour plates under aseptic conditions. The final aw of
this medium is 0.955. DG18 agar is used as a general purpose mold enumeration medium
and is preferred when the aw of the analyzed food is less than 0.95. The low water activity of
this medium reduces interference by bacteria and fast-growing fungi. When both yeasts and
molds must be enumerated, DRBC agar should be used.
Mix ingredients, heat to dissolve agar and sterilize by autoclaving at 121°C for 15 min. Temper
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Notes: DRBC agar is especially useful for analyzing samples containing "spreader" molds
(e.g. Mucor, Rhizopus, etc.), since the added dichloran and rose bengal effectively slow down
the growth of fast-growing fungi, thus readily allowing detection of other yeast and mold
propagules, which have lower growth rates.
Media containing rose bengal are light-sensitive; relatively short exposure to light will
result in the formation of inhibitory compounds. Keep these media in a dark, cool place until
used. DRBC agar should be used for spread plates only.
Mix ingredients, steam to dissolve agar and sterilize for 15 min at 121° C. Temper medium to
45° C and pour plates under aseptic conditions. To prepare slants dispense 5-6 ml of steamed
medium (before autoclaving) into each of several 16 × 125 mm screw-cap tubes, loosely cap
tubes and sterilize as above. After autoclaving lay tubes in a slanting position and let them
cool. This medium is recommended as a general maintenance medium.
Mix ingredients, heat to dissolve agar and sterilize at 121° C for 15 min. Temper media to 45°
C and pour plates under aseptic conditions. Dehydrated MEA is commercially available, but
since more than one MEA formula exists, check for the appropriate composition. This
medium is recommended for the identification of Aspergillus and Penicillium.
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Hypertext Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter
18.
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