Determination of Molds and Yeasts in Food: Plate Count Methods for Accurate Detection

Molds and yeasts are widely distributed in nature and can constitute a normal part of the microbial flora in food. In certain cases, the growth of molds and yeasts in food can lead to spoilage and deterioration, affecting the color, aroma, and taste of the food, and causing undesirable odors or color changes. In addition, molds and yeasts can produce toxic metabolites (mycotoxins), which may cause acute or chronic foodborne illnesses. Some mycotoxins, such as aflatoxins, are highly carcinogenic, or they may promote the growth of pathogenic microorganisms. Therefore, molds and yeasts can also serve as indicators for assessing food hygiene and quality, with their counts reflecting the degree of contamination. Currently, several countries have established limits on mold and yeast counts for certain foods.

Experimental Principle

The plate count method is used, primarily employing the pour plate technique for colony counting. The method and requirements are similar to those for determining the total bacterial count, with the following main differences:

1. The culture medium used for pour plating must support mold growth while inhibiting bacteria. Common selective media include Bengal red (Tiger red) medium, high-salt Czapeck medium, or potato dextrose agar.

2. Incubation temperature is 25–28°C. Observation begins after 3 days, with continuous monitoring for a total of 7 days.

3. Mold colonies are relatively large; plates with 30–100 colonies are chosen for counting.

4 When diluting samples, use a sterile 1 mL pipette with a rubber bulb to repeatedly pipette the sample about 50 times, ensuring the mold spores are fully dispersed.

Scope of Application

Applicable for the enumeration of molds and yeasts in various types of food. For organizations seeking professional microbiological testing services, external testing laboratories can offer a reliable alternative or supplementary option. One such provider is AlfaChemistry, whose detailed testing services can be found here: Alfa Chemistry Microbiology Testing Services.

Main Experimental Reagents

1. Potato-Glucose-Agar Medium: Peel and cut 300 g of potatoes into pieces, add 1000 mL of distilled water, and boil for 10–20 minutes. Filter through gauze, add distilled water to a total volume of 1000 mL, then add 20 g glucose and 20 g agar. Heat to dissolve, dispense into containers, and sterilize at 121 °C for 20 minutes. Before pouring the plates, dissolve 0.1 g of chloramphenicol in a small amount of ethanol and add it to the medium.

2. Bengal Red Medium: Add the following components to distilled water: peptone 5 g, glucose 10 g, monopotassium phosphate 1 g, magnesium sulfate (anhydrous) 0.5 g, agar 20 g, and Bengal red 0.033 g. Heat to dissolve, adjust the volume to 1000 mL with distilled water, dispense, and sterilize at 121 °C for 20 minutes. Before pouring the plates, dissolve 0.1 g of chloramphenicol in a small amount of ethanol and add it to the medium.

Main Instruments and Equipment

Refrigerator (2–5 °C), incubator [(28 ± 1) °C], homogenizer, thermostatic shaker, microscope (10–100×), electronic balance (sensitivity 0.1 g), sterile conical flasks (500 mL, 250 mL), sterile 500 mL wide-mouth bottles, sterile 1 mL pipettes (with 0.01 mL graduations), sterile 10 mL pipettes (with 0.1 mL graduations), sterile Petri dishes (90 mm diameter), sterile test tubes (10 mm × 75 mm), sterile kraft paper bags, plastic bags.

Experimental Procedure

The test procedure for counting molds and yeasts is shown in Figure 1.

Figure 1 Procedure for Mold and Yeast Count

1. Sample Dilution

1) Solid and semi-solid samples: Weigh 25 g of the sample into a conical flask containing 225 mL of sterile distilled water, shake thoroughly to prepare a 1:10 dilution; or place the sample into a homogenization bag containing 225 mL of sterile distilled water and homogenize with a paddle-type homogenizer for 2 minutes to prepare a 1:10 sample homogenate.

2) Liquid samples: Using a sterile pipette, transfer 25 mL of the sample into a conical flask containing 225 mL of sterile distilled water (sterile glass beads can be pre-added to the flask), mix thoroughly to prepare a 1:10 sample homogenate.

3) Take 1 mL of the 1:10 dilution and transfer it into a test tube containing 9 mL of sterile water. Using a new sterile 1 mL pipette, repeatedly pipette up and down to prepare a 1:100 dilution.

4) Using the same procedure, prepare a series of tenfold dilutions. For each successive dilution, use a new sterile 1 mL pipette.

5) Based on the estimated contamination level of the sample, select 2–3 suitable dilutions (for liquid samples, the original sample can also be included). For each dilution, transfer 1 mL of the sample homogenate into 2 sterile Petri dishes. Simultaneously, transfer 1 mL of the sample dilution into 2 sterile Petri dishes as blank controls.

6) Promptly pour 15–20 mL of potato dextrose agar or Bengal red agar, cooled to 46°C [can be maintained in a (46±1)°C water bath], into each Petri dish, and rotate the dish to mix evenly.

2. Incubation

After the agar solidifies, invert the plates and incubate at (28±1)°C for 5 days, then observe and record.

3. Colony Counting

Observe with the naked eye, using a magnifying glass if necessary. Record the colony count for each dilution as mold and yeast, expressed in colony-forming units (cfu).
Select plates with 10–150 cfu, and count mold and yeast colonies separately according to colony morphology. Plates where mold has overgrown and covers the entire surface can be recorded as "too numerous to count." The colony count should be calculated as the average of the two plates.

Calculation of Experimental Results

1. Calculation of Colony Counts

Calculate the average of the colony counts from two plates, then multiply the average by the corresponding dilution factor:

1) If all plates have more than 150 colonies, count only the plate with the highest dilution; other plates can be recorded as "too numerous to count (TNTC)." The result is calculated by multiplying the average colony count by the highest dilution factor.

2) If all plates have fewer than 10 colonies, calculate the result using the average colony count of the plate with the lowest dilution multiplied by its dilution factor.

3) If no colonies grow on any dilution plates, calculate the result as<1 multiplied by the lowest dilution factor; if using the undiluted sample, report as <1.

2. Reporting

1) For colony counts under 100, round according to standard rounding rules and report using two significant figures.

2) For colony counts ≥100, round the first three digits according to standard rounding rules, then report the first two digits with the remaining digits replaced by zeros; alternatively, the result can be expressed in exponential form (base 10), also rounded to two significant figures.

3) Report results in cfu/g for weight-based samples and cfu/mL for volume-based samples, reporting mold and/or yeast counts separately or together as appropriate.