SOP: Enumeration of Lactic Acid Bacteria in Foods by the Pour Plate Method

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This SOP enumerates lactic acid bacteria in foods following CCFRA Chapter 14.1. Measured volumes of the sample dilution are mixed with MRS agar poured in two layers. The plates are incubated under anaerobic conditions at 30 °C ± 1 °C for 72 ± 4 hours; the colonies are then counted and the count is calculated as colony-forming units per gram or millilitre of the food.

2. Apparatus required

• Sterile Petri dishes
• Sterile pipettes
• Water bath, for tempering the agar
• Incubator capable of maintaining 30 °C ± 1 °C
• Anaerobic jar with a gas-generating system

3. Reagents

• A. MRS agar, poured as a double layer
• To prepare: use commercially available lactobacilli MRS broth and add 15.0 g of agar per L of broth

4. Sample preparation

• Prepare a 1 in 10 dilution of the sample and further decimal dilutions.

5. Procedure

Label the plates with the sample and the dilution
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Pipette 1 mL of the dilution into each of two sterile Petri dishes
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Pour a layer of tempered MRS agar into each dish and mix gently
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Allow the agar to solidify, then pour a second layer of MRS agar over the surface
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Allow the second layer to solidify
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Place the plates in the anaerobic jar and establish anaerobic conditions
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Incubate at 30 °C ± 1 °C for 72 ± 4 hours
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Count the colonies on each plate

Alternative set-ups: pour 10 mL over 3 plates (3 mL, 3 mL and 4 mL), or use 2 mL per dilution.

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 (2 mL for the 1 mL in duplicate scheme; 10 mL for the 10 mL over 3 plates scheme)
• D = dilution of the plated dilution (for example, 10^−1)

Report the count as colony-forming units per gram or millilitre of the food.

7. Precautions

• Keep the plates under anaerobic conditions for the whole incubation period.
• Count the plates at the end of the stated incubation time.

8. References

• CCFRA, Chapter 14.1 — enumeration of lactic acid bacteria.