Enumeration of Listeria monocytogenes in foods

Help on accessing alternative formats, such as Portable Document Format (PDF), Microsoft Word and PowerPoint (PPT) files, can be obtained in the alternate format help section.

Laboratory Procedure MFLP-74
February 2011

Health Products and Food Branch

Ottawa

Enumeration of Listeria monocytogenes in Foods

Franco Pagotto¹, Yvon-Louis Trottier¹, Jacqueline Upham² and Irene Iugovaz¹

¹ Research Division, Bureau of Microbial Hazards, Food Directorate, Health Canada, Ottawa, Ontario, HPFB, Postal Locator 2204E, K1A 0K9
² Canadian Food Inspection Agency, Microbiology, Dartmouth Laboratory, 1992 Agency drive, Dartmouth, NS, B3B 1Y9.

E-mail: micro_methods_committee@hc-sc.gc.ca

1. Application

This method is applicable to the enumeration of viable Listeria monocytogenes to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. This method replaces MFLP-74, dated August 2010.

2. Principle

This direct plating procedure quantitatively determines the number of viable Listeria monocytogenes in the product. A portion of the product is blended in a suitable diluent, surface-plated onto at least two selective agars, and the plates are then incubated under specified conditions of time and temperature. It is assumed that each viable cell of L. monocytogenes will multiply under these specified incubation conditions and give rise to a visible colony which can be identified. The selective media used are based on studies done by other investigators (7.3, 7.4, 7.5).

3. Definition of terms

See Appendix A of Volume 3.

4. Collection of samples

See Appendix B of Volume 3.

5. Materials and special equipment

See MFHPB-30, 'Materials and Special Equipment'.

See Appendix G for the formulas of individual media

Additional requirements:

1) Peptone water, 0.1% (w/v)

2) Plating media:
Selective media include the following:

OXA (mandatory agar)

One of the following is to be included with OXA:

• - Agar Listeria according to Ottaviani & Agosti
• - A.L. agar
• - BBL Chromagar Listeria
• - Brilliance Listeria Agar (formerly OCLA agar)
• - Lithium chloride-phenylathanol-moxalactam medium (LPM)
• - Modified Oxford agar (MOX)
• - Palcam agar (PAL)
• - Rapid L'Mono

6. Procedure

Information regarding the distribution of Listeria monocytogenes can be obtained by testing each analytical unit separately.

6.1 Handling and Dilution of Sample Units

6.1.1 In the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated or frozen depending on the nature of the product. Thaw frozen samples in the refrigerator, or under time and temperature conditions which prevent microbial growth or death.

6.1.2 Analyses should be started as soon as possible after receipt of samples in the laboratory.

6.1.3 To ensure a representative analytical unit, agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. The analytical sample recommended is 10 grams or 10 ml.

6.2 Direct Plating Procedure

6.2.1 Prepare a 1:5 dilution of the sample, as required, in 0.1% (w/v) peptone water in a blender jar or stomacher bag. Blend or stomach for 2 minutes to ensure thorough mixing. For food matrices that require a higher dilution to allow for ease of spreading the food/diluent slurry on the selective plates, a 1:10 dilution may be used. This may require the plating of additional sample aliquot over additional plates.

Two agar types are used to enumerate L. monocytogenes as follows: immediately spread 1 ml by taking three, 0.333 mL portions of the diluted food, and spread onto the surface of 3 plates of each selective media (e.g., a total of 2 mL is plated over two agar types). Selective media includes OXA and one of the approved agars listed in section 5.

6.2.2 Incubate LPM plates at 30°C and all other selective agars at 35°C for 48 h, unless the time and temperature are otherwise directed by the manufacturer. Examine plates at 24 h for typical colonies, as well as at 48 h as applicable to the medium. Observe for colonies with a typical appearance (section 6.3.1 to 6.3.8).

6.3 Presumptive Identification

6.3.1 OXA and MOX agars - L. monocytogenes forms 1 mm diameter black colonies surrounded by black haloes after 24 h. At 48 h, colonies are 2-3 mm in diameter, black with a black halo and sunken centre. The colonies can also appear brown-black and green-black. Other Listeria spp. shows a similar appearance. When examined before 24 h, growth of Listeria spp. is sometimes apparent but without the characteristic blackening. Some strains of this genus other than L. monocytogenes are inhibited on this medium when incubated at 35°C.

6.3.2 LPM - Examine LPM plates for suspect colonies using beamed white light powerful enough to illuminate the plate well, striking the plate bottom at a 45° angle (7.6). Under optimum transillumination, the more isolated and larger (48 h old) Listeria spp. colonies appear as whitish piles of crushed glass often showing mosaic-like internal structures, occasionally having blue-grey iridescence that tends to sparkle. Alternatively, the colonies can look smooth with a blue tinge around the perimeter. When growth becomes near confluent, an even blue-grey iridescent sheen can be observed.

6.3.3 PAL agar - L. monocytogenes colonies are approximately 2 mm grey-green with a black sunken centre and a black halo on a cherry-red background. Some Enterococcus and Staphylococcus strains produce grey colonies with a brown-green halo or yellow colonies with a yellow halo.

6.3.4 Agar Listeria according to Ottaviani & Agosti - Listeria colonies appear blue-green, with L. monocytogenes and L. ivanovii colonies having opaque halos surrounding the colonies after 24 h. All other Listeria species are blue-green but do NOT have the halo. Consult manufacturer insert for a more detailed description.

6.3.5 A.L. agar - All Listeria species form blue to blue-green colonies, with L. monocytogenes and L. ivanovii colonies having opaque halos surrounding the colonies after 24 and 48 h, respectively.

6.3.6 BBL CHROMagar - L. monocytogenes and L. ivanovii appear as blue-green colonies surrounded by a opaque white halo. Other Listeria spp. are blue-green colonies without a halo.

6.3.7 Brilliance Listeria Agar (formerly OCLA agar) - L. monocytogenes and L. ivanovii appear as blue colonies surrounded by an opaque halo, whilst other Listeria species produce blue colonies without a halo after 24 h.

6.3.8 Rapid L'Mono agar - L. monocytogenes forms blue colonies without a yellow halo whereas L. ivanovii are greenish-blue with a yellow halo. All other Listeria species are yellow to white in colour.

6.4 Enumeration and Confirmation of Colonies from Direct Plating Procedure

6.4.1 Examine each set of triplicate plates from the two selective media used, for typical colonies. Because it is expected that listeriae will be present in low numbers, each colony must be confirmed prior to reporting of the results, up to 15 colonies per agar type.

6.4.2 Confirm presumptive colonies as L. monocytogenes by following the procedures described in MFHPB-30.

6.5 Interpretation

6.5.1 Record the results separately for each of the two selective agars. Calculate the colony forming units (CFU) per g (or ml) using the following equation:

Number of CFU / Volume plated (ml) x total dilution factor

Example:

A 1:5 dilution is made from a food matrix. One (1) ml is spread onto three OXA plates and 1 ml is spread onto three PAL plates. After incubation, the sum total of each OXA plate is 5 colonies and the sum total of the PAL plates is 15 colonies.

Using the above equation:

5 colonies / 1 (volume plated, ml) x 0.2 (dilution factor)

= 25 CFU/g

Repeating with the PAL plates:

15 colonies / 1 (volume plated, ml) x 0.2 (dilution factor)

= 75 CFU/g

Final report = 75 CFU/g [Note that only the highest number is reported]

7. References

7.1 Pagotto, F. Daley, E., and Farber, J.M. 2002. Enumeration of Listeria monocytogenes in foods (MLFP-74). The Compendium of Analytical Methods, Volume 3. Available at http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume3/mflp74-eng.php.

7.2 MLFP-74 Supplement. 2004. The Compendium of Analytical Methods, Volume 3. Available at http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume3/mflp74-sup-eng.php.

7.3 Poysky, F.T., Paranjpye, R.N., Lashbrook, L.C., Peterson, M.E., Pelroy, G.A., and Eklund, M.W. 1993. Selective and differential medium for isolation of Listeria monocytogenes from foods. Journal of Food Protection 56:326-332.

7.4 Warburton, D.W., Farber, J.M., Armstrong, A., Caldeira, R., Hunt, T., Messier, S., Plante, R., Tiwari, N.P., and Vinet, J.1991. A comparative study of the "FDA" and "USDA" methods for the detection of Listeria monocytogenes in foods. International Journal of Food Microbiology 13:105-118.

7.5 Warburton, D.W., Farber, J.M., Armstrong, A., Caldeira, R., Tiwari, N.P., Babiuk, T., LaCasse, P., and Read, S. 1991. A Canadian comparative study of modified versions of the "FDA" and "USDA" methods for the detection of Listeria monocytogenes. Journal of Food Protection 54:669-676.

7.6 American Public Health Association (APHA). 2001. Compendium of Methods for the Microbiological Examination of Foods. Fourth edition. Chapter 35. F.P. Downes and K. Ito (editors), American Public Health Association, Washington, D.C.

7.7 Pagotto, F., Hébert, K., J. Farber. 2011. Isolation of Listeria monocytogenes and other Listeria spp. from foods and environmental samples. In: Volume 2. The Compendium of Analytical Methods. http://www.hc-sc.gc.ca/food-aliment.

Figure 1: Generic diagram of enumeration procedure

End of Document