Commission Decision of 5 February 2010 concerning a financial contribution from the Union towards a coordinated monitoring programme on the prevalence of Listeria monocytogenes in certain ready-to-eat foods to be carried out in the Member States (notified under document C(2010) 592) (2010/75/EU)

2. Sample preparation and initial suspension preparation

Cross contamination between samples and from the surrounding environment shall be avoided at all stages. Samples are discarded once laboratory analyses have been initiated. If the analysis is stopped, for example due to unacceptable deviations in the analysis process, new samples must be obtained.

Either the entire product, or a representative test portion of 100 to 150 g, shall be taken to the initial dilution. Food shall be sampled to include surfaces reflecting the proportion that would be consumed (such as 20 % rind/surface and 80 % inside). When a packaged product is sliced, the respective sample is taken from more than one slice of the product. The test portion shall be cut in small pieces and placed into a stomacher bag, using a sterile instrument and an aseptic technique. From that mixture, a test portion of 10 g shall be taken for enumeration and a test portion of 25 g shall be taken for detection.

To the volume of the test portion (10 g), 9 volumes (90 ml) of diluent are added and subsequently the mixture is homogenized using a stomacher or a pulsifier for 1 to 2 min.

Buffered peptone water, as described in EN ISO 11290-2 ‘Microbiology of food and animal feeding stuffs — Horizontal method for detection and enumeration of Listeria monocytogenes — Part 2: Colony-count technique’, may be applied as a diluent for general use.

For the dilution of cheese, a sodium citrate solution, as described in EN ISO 6887-5 ‘Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 5: Specific rules for the preparation of milk and milk products’ shall be used.

Detection and enumeration analyses of Listeria monocytogenes shall be performed in accordance with the following:

(a)

for smoked and gravad fish samples two sets of analyses must be carried out:

(i)

immediately after sample collection at retail level and

(ii)

at the end of shelf-life;

(b)

for soft and semi-soft cheese samples and heat-treated meat product samples the analyses must be carried out only at the end of shelf-life.

2.1. Detection of Listeria monocytogenes

Detection of Listeria monocytogenes shall be performed according to the amended version of EN ISO 11290-1:1996 ‘Microbiology of food and animal feeding stuffs — Horizontal method for the detection and enumeration of Listeria monocytogenes — Part 1: Detection method’.

2.2. Enumeration of Listeria monocytogenes

The enumeration of Listeria monocytogenes shall be performed according to EN ISO 11290-2:1998 ‘Microbiology of food and animal feeding stuffs — Horizontal method for the detection and enumeration of Listeria monocytogenes — Part 2: Enumeration method’ and its modification EN ISO 11290-2:1998/Amd 1:2004 ‘Modification of the enumeration medium’.

If the sample is found to be contaminated, it is assumed that the majority of products would contain low contamination levels of Listeria monocytogenes. To enable the estimation of low numbers in samples (between 10 and 100 cfu/g), 1 ml of the primary dilution shall be tested in duplicate as indicated in EN ISO 11290-2:1998/Amd 1:2004:

(a)

spread onto the surface of three 90-mm diameter plates, or

(b)

spread onto the surface of one 140-mm diameter plate.

Because of the possibility of higher contamination levels of Listeria monocytogenes, 0,1 ml of the primary dilution must be spread onto the surface of one plate to allow the enumeration of up to 1,5 × 10⁴ cfu/g. This plating must be performed in single as provided in ISO 7218:2007 ‘Microbiology of food and animal feeding stuffs — General requirements and guidance for microbiological examinations’.