3.EXAMINATION OF THE SAMPLES
3.1. Transport and preparation of the samples
Samples shall preferably be sent by express mail or courier to the laboratories referred to in Articles 11 and 12 of Regulation (EC) No 2160/2003, within 24 hours after collection. If they are not sent within 24 hours, they shall be stored refrigerated. The samples may be transported at ambient temperature provided that excessive heat (namely over 25 °C) or exposure to sunlight is avoided. At the laboratory the samples shall be kept refrigerated until examination, which must be started within 48 hours following receipt and within 4 days after sampling.
Separate preparations shall be made of the boot swabs and dust or the fabric dust swab in the case of samples by the competent authority, but as regards samples by food business operators the different sample types may be combined in one test.
3.1.1. Boot and fabric swab samples
(a)The two pairs of boot swabs (or ‘socks’) or dust swabs shall be carefully unpacked to avoid dislodging adherent faecal material, pooled and placed in 225 ml of Buffered Peptone Water (BPW) which has been pre-warmed to room temperature, or the 225 ml of diluent must be added directly to the two pairs of boot swabs in their container as received in the laboratory. The boot/socks or fabric swab shall be fully submersed in BPW to provide sufficient free liquid around the sample for migration of Salmonella away from the sample and therefore more BPW may be added if necessary.
(b)The sample shall be swirled to fully saturate it and culture shall be continued by using the detection method set out in point 3.2.
3.1.2. Other faecal and dust material
(a)The faeces samples shall be pooled and thoroughly mixed and a 25-gram sub-sample shall be collected for the culture.
(b)The 25-gram sub-sample (or 50 ml of suspension containing 25 grams of the initial sample) shall be added to 225 ml of BPW which has been pre-warmed to room temperature.
(c)Culture of the sample shall be continued by using the detection method set out in point 3.2.
If ISO standards on the preparation of relevant samples for the detection of Salmonella are agreed on, they shall be applied and replace those set out in points 3.1.1 and 3.1.2.
3.2. Detection method
The detection of Salmonella shall be carried out according to Amendment 1 of EN/ISO 6579-2002/Amd1:2007 ‘Microbiology of food and animal feeding stuffs – Horizontal method for the detection of Salmonella spp. – Amendment 1: Annex D: Detection of Salmonella spp. in animal faeces and in environmental samples from the primary production stage’ of the International Organization for Standardization.
After incubation the samples in BPW shall not be shaken, swirled or otherwise agitated.
3.3. Serotyping
At least one isolate from each positive sample taken by the competent authority shall be serotyped, following the Kaufmann-White-LeMinor scheme. In isolates taken by the food business operators, at least the serotyping for Salmonella Enteritidis and Salmonella Typhimurium must be carried out.
3.4. Alternative methods
With regard to samples taken on the initiative of the food business operator, alternative methods may be used instead of the methods for the preparation of samples, detection methods and serotyping set out in points 3.1, 3.2 and 3.3 of this Annex, if validated in accordance with the most recent version of EN/ISO 16140.
3.5. Testing for antimicrobial resistance
The isolates shall be tested for antimicrobial resistance in accordance with Article 2 of Commission Decision 2007/407/EC().
3.6. Storage of strains
The competent authority shall ensure that at least one isolated strain of the relevant Salmonella serotypes from sampling as part of official controls per house and per year is stored for possible future phagetyping or antimicrobial susceptibility testing, using the normal methods for culture collection, which must ensure integrity of the strains for a minimum period of 2 years.
If the competent authority so decides, isolates from sampling by food business operators shall also be stored for these purposes.