SOP: Yeast and Mold Count
GIFSQ — Global Institute for Food Safety & Quality
1. Principle
A culture medium carrying enough nutrients for growth of most yeasts and moulds, together with antibiotics that suppress most bacteria, is inoculated with a measured quantity of the product and incubated; colonies are counted after 5 days at 22 to 25 °C. The count, in colony-forming units per gram or millilitre, is reported for yeasts and for moulds. This is a general-purpose method and may not detect yeasts and moulds adapted to certain foods, such as foods of low water activity.
2. Apparatus required
- Sterile rods (spreaders)
- Stomacher, blender or equivalent
- Colony counting device (optional)
- Incubator capable of maintaining 22 to 25 °C
- Water bath at 45 °C, as needed to temper agar and/or to pre-warm sodium citrate
3. Media and reagents
All media and reagents listed are commercially available and are used, prepared and/or sterilized according to the manufacturer’s instructions.
Diluents
- Peptone water, 0.1% (PW) — the routine diluent for most foods
- To prepare: suspend 1.0 g of peptone in 1.0 L of distilled water; mix thoroughly and dispense; autoclave at 121 °C for 15 min. Final pH 7.0 ± 0.2.
- Peptone water (0.1%) with 20% sucrose (W/V) — diluent for high-sugar foods such as honey
- To prepare: dissolve 1.0 g of peptone and 200 g of sucrose in distilled water and make up to 1.0 L; autoclave at 121 °C for 15 min.
- 2% sodium citrate pre-warmed to 45 °C — diluent for high-fat foods such as cheese
- To prepare: dissolve 20.0 g of trisodium citrate dihydrate in 1.0 L of distilled water by heating to 45–50 °C; adjust the pH to 7.5 ± 0.2; dispense; autoclave at 121 °C for 15 min.
Potato dextrose agar with chloramphenicol (PDA-C)
- Basal medium: potato dextrose agar — 1.0 L (potatoes — 200.0 g; dextrose — 20.0 g; agar — 20.0 g per litre of distilled water); chloramphenicol — 100.0 mg. Final pH 5.6 ± 0.2.
- To prepare: prepare 1.0 L of potato dextrose agar and add 100.0 mg of chloramphenicol ↓ mix thoroughly and autoclave at 121 °C for 15 min ↓ dispense into sterile Petri dishes.
Dichloran Rose Bengal chloramphenicol agar (DRBC)
- Basal medium (per 990 mL): glucose — 10; peptone — 5.0 g; potassium phosphate, monobasic (KH2PO4) — 1.0 g; magnesium sulfate, 7 hydrate (MgSO4·7H2O) — 0.5 g; Rose Bengal — 0.025 g; dichloran — 0.002 g; agar — 15.0 g; distilled water — 990 mL. pH 5.6 ± 0.2. Chloramphenicol supplement: chloramphenicol — 0.1 g in 10.0 mL of distilled water (filter-sterilized).
- To prepare: add the components, except the chloramphenicol solution, to distilled water and bring the volume to 990 mL ↓ mix thoroughly ↓ gently heat and bring to a boil ↓ autoclave at 121 °C for 15 min ↓ aseptically add the filter-sterilized chloramphenicol solution and mix thoroughly ↓ pour into plates.
4. Preparation of sample
Keep sample units refrigerated or frozen in the laboratory before analysis, depending on the nature of the product; shelf-stable foods are the exception
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Thaw frozen samples in a refrigerator, or under time and temperature conditions that prevent microbial growth or death
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Analyse the sample units as soon as possible after receipt
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Choose the diluent: 0.1% peptone water for most foods; 2% sodium citrate pre-warmed to 45 °C for high-fat foods including cheese; 0.1% peptone water with 20% sucrose for high-sugar foods such as honey
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Agitate liquids or free-flowing materials until homogeneous; for a solid sample unit, take portions from several locations within the unit to make a truly representative analytical unit
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For dried or intermediate-moisture foods, re-hydrate the dried food for 30 to 60 minutes with an equal amount of peptone water at room temperature — soaking helps recover yeasts and moulds and repair sub-lethally damaged cells
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Prepare a 1 in 10 dilution of the food: aseptically weigh 25 g or mL (the analytical unit) into 225 mL of the chosen diluent. If a dried food was soaked, prepare a 1 in 5 dilution of that 1 in 2 dilution, giving a 1 in 10 dilution overall. If a different sample size is used, keep the 1 in 10 sample-to-diluent ratio, such as 11 (10) g or mL into 99 (90) mL
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Blend, stomach or vortex as needed for thorough mixing
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Prepare succeeding decimal dilutions as required
5. Procedure
There are three separate ways to plate this method — a PDA spread plate, a PDA pour plate, and a DRBC spread plate. They are three variants of one method, not three methods. Pick the variant the laboratory requires and follow it alone.
Variant 1 — PDA spread plate
Pour the prepared PDA-C into sterile Petri dishes and let the agar set
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Before transfers, agitate any dilution bottle that shows signs of material settling out, and any bottle that has rested for more than 5 minutes
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Spread 0.1 mL, in duplicate from each dilution, onto the PDA-C plates; to help enumerate low yeast populations, larger volumes such as 0.25 mL of the 10⁰ or 10⁻¹ dilutions may be used
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Incubate the plates undisturbed in the upright position at 22 to 25 °C for 5 days
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Examine the plates on the third day; if mould colonies are numerous, count them and re-incubate for a total of 5 days — handle the plates as little as possible during the day-3 count so spores are not dislodged, which would cause secondary growth
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Count the plates again on day 5; if plates are TNTC on day 5, report the day-3 counts; otherwise report the day-5 counts
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Count the colonies, distinguishing yeast colonies from mould colonies by colonial morphology: yeasts appear as smooth creamy colonies, moulds as fuzzy filamentous colonies
Variant 2 — PDA pour plate
Use pour plates for yeasts or non-stressed mould cells; for moulds stressed by drying, heating, chilling or freezing, use the surface spread-plate technique (Variant 1) instead — it gives the cells maximal exposure to atmospheric oxygen and avoids heat stress from molten agar
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Pipette 1 mL or 0.1 mL of the required dilutions into appropriately marked duplicate Petri plates
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Pour at least 15 mL of tempered PDA-C agar into each plate and mix by rotating and/or tilting
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Let the plates solidify; pour the plates not more than 15 minutes after the dilutions were prepared
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Incubate the plates undisturbed in the upright position at 22 to 25 °C for 5 days
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Examine the plates on the third day; if mould colonies are numerous, count them and re-incubate for a total of 5 days — handle the plates as little as possible during the day-3 count so spores are not dislodged, which would cause secondary growth
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Count the plates again on day 5; if plates are TNTC on day 5, report the day-3 counts; otherwise report the day-5 counts
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Count the colonies, distinguishing yeast colonies from mould colonies by colonial morphology: yeasts appear as smooth creamy colonies, moulds as fuzzy filamentous colonies
Variant 3 — DRBC spread plate
DRBC is the separate spread-plate variant for samples where spreading moulds make counting difficult: the rose bengal and dichloran in the medium restrict spreading moulds, making counting easier. Use it when the PDA plates are overrun by spreaders
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Before transfers, agitate any dilution bottle that shows signs of material settling out, and any bottle that has rested for more than 5 minutes
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Spread 0.1 mL, in duplicate from each dilution, onto the DRBC plates
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Incubate the plates undisturbed in the upright position at 22 to 25 °C for 5 days
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Do not expose DRBC agar to light during handling and incubation: photo-degradation of Rose Bengal produces compounds toxic to fungi
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Examine the plates on the third day; if mould colonies are numerous, count them and re-incubate for a total of 5 days — handle the plates as little as possible during the day-3 count so spores are not dislodged, which would cause secondary growth
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Count the plates again on day 5; if plates are TNTC on day 5, report the day-3 counts; otherwise report the day-5 counts
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Count the colonies, distinguishing yeast colonies from mould colonies by colonial morphology: yeasts appear as smooth creamy colonies, moulds as fuzzy filamentous colonies
Selecting countable plates
Where possible, select plates with 10 to 150 colonies; if counts fall outside this range, take the plates nearest to the 10–150 range and report as an estimated count — where the mycoflora is mainly moulds use the lower counting range, where it is mainly yeasts use the upper limit
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If plates carry spreaders, count a representative spreader-free portion of the plate if possible; estimate the whole-plate count by multiplying the portion count by the reciprocal of the fraction of the plate counted — for example, 30 colonies on 1/4 of the plate gives 30 × 4 = 120 colonies; report as an estimated count
6. Calculation
- N = A × D
Where:
- N = number of colonies (yeasts and moulds) per g (mL) of product
- A = average colony count per plate
- D = dilution factor (the reciprocal of the dilution)
Round counts to two significant figures, to the nearest whole number.
If the lowest dilution plated shows no colonies, report the lowest average obtainable with the volume plated on the set of replicate plates, preceded by a “less than” (<) sign: for 1 mL on a set of duplicate plates (1 mL per plate), the value is <0.5 — the lowest possible average with one colony on one of the two plates is (1 + 0) ÷ 2 = 0.5 for a 10⁰ dilution (dilution factor = 1). For other dilutions, multiply 0.5 by the reciprocal of the dilution (the dilution factor): if 2 mL of the 10⁻¹ dilution is plated (pour or spread), the reported value with no yeasts or moulds present is <5 cfu/g; if 0.2 mL is plated by the spread method, it is <50 cfu/g.
7. Precautions
- Some yeasts and moulds can be infectious or can cause allergic responses — be cautious when working with fungi.
- Ideally keep plates in incubators, not in an open room.
- Remove plate lids only when needed, for example to prepare a slide for microscopic examination.
- Cool flamed needles before making transfers, to avoid dispersing conidia and other cells.
- Avoid inhaling spores and other fungal products such as volatiles — do not smell plates.
- Do not expose DRBC agar to light: photo-degradation of Rose Bengal produces compounds toxic to fungi.
8. References
Health Canada, Compendium of Analytical Methods, Volume 2 — HPB Method MFHPB-22, April 2018: “Enumeration of Yeasts and Moulds in Foods” (replaces MFHPB-22, January 2004).