SOP: Enumeration of Osmophilic and Xerophilic Yeasts and Moulds (MY40G, MY50G and DG18)

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This SOP enumerates osmophilic and xerophilic yeasts and moulds in foods with a low water activity following the APHA Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapters 17 and 20, 4th Edition. Organisms that grow at high sugar concentrations do not have an absolute requirement for a reduced water activity but tolerate drier environments better than other species; yeasts are the most common osmophilic spoilage organisms of high-sugar foods such as jams, honey, concentrated fruit juices, molasses and syrups. Because hypotonic diluents can destroy osmophilic yeasts, the diluents also carry a reduced water activity. Three media variants are used: MY40G as the general-purpose medium for most osmotolerant microorganisms, MY50G for extreme xerophiles, and DG18 for moderate xerophiles in low-water-activity foods such as cereals, flours, nuts and spices. Measured volumes of the sample dilution are plated, incubated, and the colonies are counted and calculated as colony-forming units per gram or millilitre of the food.

2. Apparatus required

• Stomacher or blender
• Sterile Petri dishes, 15 × 100 mm
• Sterile pipettes
• Sterile bent glass rod
• Stainless steel filter apparatus
• Type HA filters with grid marks and absorbent pads
• Vacuum pump and filter flask, 1 L
• Erlenmeyer flask, 500 mL, screw cap
• Incubator capable of maintaining 25 °C
• Incubator capable of maintaining 30 °C
• Colony counter

3. Reagents

• A. Malt extract yeast extract 40% glucose agar (MY40G): malt extract powder — 12.0 g; yeast extract — 3.0 g; agar — 12.0 g; distilled water to make — 600.0 g; glucose, AR — 400.0 g
• To prepare: combine the ingredients except the glucose with 550.0 mL of distilled water and steam to dissolve the agar ↓ immediately make up to 600.0 g with distilled water ↓ while the solution is still hot, add the glucose all at once and stir rapidly to prevent the formation of hard lumps of glucose monohydrate (if lumps form, dissolve them by steaming for a few minutes) ↓ steam the medium for 30 min
• This medium is of sufficiently low water activity (a_w near 0.92, final pH about 5.5) not to require autoclaving
• B. Malt extract yeast extract 50% glucose agar (MY50G): malt extract powder — 10.0 g; yeast extract — 2.5 g; agar — 10.0 g; distilled water — 0.5 L; glucose monohydrate — 500.0 g
• To prepare: combine the first three ingredients with 450 mL of distilled water and steam to dissolve ↓ make up to 500 mL with distilled water ↓ add the glucose and stir rapidly to prevent lump formation ↓ steam for 30 min
• The final water activity is 0.89; autoclaving is not necessary
• C. Dichloran 18% glycerol agar (DG18): peptone — 5.0 g; glucose — 10.0 g; potassium phosphate, monobasic (KH₂PO₄) — 1.0 g; magnesium sulfate, 7 hydrate (MgSO₄·7H₂O) — 0.5 g; dichloran (0.2% in ethanol) — 1.0 mL; chloramphenicol — 0.1 g; glycerol — 220.0 g; agar — 20.0 g; distilled water — 1.0 L
• To prepare: add the components, except the glycerol, to 800 mL of distilled water ↓ steam to dissolve, then add water to 1.0 L ↓ add the glycerol and autoclave at 121 °C for 15 min ↓ final pH 5.6 ± 0.2
• D. High-sugar diluent: sterile phosphate-buffered water supplemented with 40% glucose, or 30% glycerol
• To prepare the 40% glucose diluent: dissolve 40 g of glucose in distilled water and make up to 100 g total (40% w/w); sterilize by steaming at 100 °C for 30 min
• To prepare the 30% glycerol diluent: add 30 mL of glycerol to distilled water and make up to 100 mL
• Glucose or invert sugar is the most suitable hexose; sucrose and corn syrup at reasonable concentrations are unlikely to give a sufficiently low water activity

4. Sample preparation

• Collect samples in sterile containers and store them in conditions that will not promote the growth of viable fungi; analyse the samples the same day they are collected.
• Stomach or blend the samples and prepare the dilutions with the high-sugar diluent so that the cells are not subjected to osmotic shock.

5. Procedure

Variant 1 — MY40G plate count (general-purpose, most osmotolerant microorganisms)

Stomach or blend the sample
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Prepare dilutions with sterile phosphate-buffered water supplemented with glucose or another solute
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Inoculate 1 mL of the desired dilution into Petri dishes, then pour with 15 to 20 mL of MY40G agar
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Swirl the plates well to distribute the sample uniformly through the viscous agar
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Incubate the plates at 30 °C for 5 to 7 days
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Count the colonies with a colony counter; many osmophilic yeasts grow slowly and their colonies are difficult to see with the unaided eye

Membrane filter technique for low numbers in viscous, low-a_w products
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Weigh a 25-g sample aseptically into a sterile Erlenmeyer flask and add 1 to 2 volumes of sterile distilled water
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Filter the sample through a sterile HA filter in the filter apparatus; rinse the flask and funnel walls with sterile water
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Remove the filter and place it, grid side up, on MY40G agar
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Incubate the dish for 5 days at 30 °C and count the colonies

Variant 2 — MY50G (extreme xerophiles)

Prepare the sample and the dilutions as in Variant 1, using the 40% glucose or 30% glycerol diluent for high-sugar foods to minimize osmotic shock
↓
Inoculate 0.1 mL of appropriate decimal dilutions in duplicate on solidified MY50G agar and spread over the entire surface with the sterile bent glass rod
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Incubate the upright plates at 25 °C for 7 days; extreme xerophiles may take up to 3 weeks to grow
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Count the plates containing 15 to 150 colonies and report the count as CFU per g or mL of sample

Variant 3 — DG18 (moderate xerophiles in low-water-activity foods)

Prepare the sample and the dilutions as in Variant 1
↓
Inoculate 0.1 mL of appropriate decimal dilutions in duplicate on solidified DG18 agar and spread over the entire surface with the sterile bent glass rod
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Incubate the upright plates at 25 °C for 7 days; the plates do not need to be stored in the dark
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Count the plates containing 15 to 150 colonies and report the count as CFU per g or mL of sample

6. Calculation

CFU per g or mL = N / (V × D)

Where,

• N = total number of colonies counted on all plates of the dilution
• V = total volume of diluted sample plated, in mL
• D = dilution of the plated dilution (for example, 10⁻¹)

Counts of 10 or fewer per g in products such as liquid sugars or syrups may be highly significant if the yeast is a spoilage type such as Zygosaccharomyces rouxii; acceptable levels are product dependent.

7. Precautions

• Sterilization of high-sugar media by autoclaving can produce compounds that are toxic to osmophiles; steam media containing high sugar at 100 °C for 30 min instead.
• Because the plating media are very viscous, swirl the plates more than usual to ensure the sample is uniformly distributed through the agar.
• Do not incubate plates with low-a_w media in a forced-draft incubator; the plates will dry and colonies will not develop fully — place a shallow pan of distilled water in the bottom of the incubator to prevent drying.
• Do not use hypotonic diluents; osmophilic yeasts may be destroyed by them.
• Diluting samples with distilled water for the membrane filter technique may cause osmotic stress; keep the analysis rapid.
• Do not smell the cultures; before disposal, autoclave the plates and other cultures.

8. References

• APHA, Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapters 17 and 20, 4th Edition.