Food and animal feeding stuffs – Horizontal method for the detection and enumeration of Listeria monocytogenes – Detection method AS 5013.24.1-2009

This standard is an adoption with national modifications and reproduced from ISO 11290:1996, Microbiology of food and animals feeding stuffs – Horizontal method for the detection and enumeration of Listeria monocytogenes, Part 1: Detection method including Amendment 1:2004.

SCOPE Products intended for human consumption or animal feeding

PRINCIPLES Detection of Listeria monocytogenes can be broken down into four successive stages:

. Primary enrichment in half-strength selective liquid enrichment medium Sample is dilute 1:10 using half Fraser broth. The initial suspension is incubated at 30C for 24 ± 3 h. Blackening of half Fraser broth indicates the possible presence of Listeria spp. Nevertheless all samples irrespective of blackening in half Fraser broth must be carried through to the next stage.

. Secondary enrichment in full-strength selective liquid enrichment medium A portion of the incubated primary enrichment culture (0.1 ml) is inoculated into 10 ml of Fraser broth. Secondary enrichment broth is incubated at 37C for 48 ± 3 h. All samples irrespective of blackening in Fraser broth must be carried through to the next stage.

. Plating out and identification Primary and secondary enrichment cultures (incubated for 24 h and 48 h, respectively) are streaked onto two plating media, ALOA1 and PALCAM or Oxford agars2. ALOA plates are incubated at 37C initially for 24 ± 3 h and then for a further 24 ± 3 h if growth is slight or if no colonies are observed after 24 h. The second selective media is incubated as per the manufacturers’ instructions. Typical Listeria colonies on ALOA agar are green-blue colonies surrounded by an opaque halo (typical colony) although some strains of L. monocytogenes show a very weak or no halo. On PALCAM colonies appear small, greyish green in colour with black halos and sometimes black centres. After longer incubation colonies appear green with a central depression surrounded by a black halo. On Oxford Listeria colonies are small (1 mm) greyish and surrounded by a black halo. After longer incubation colonies are larger and darker, with possibly a greenish colour, black halos and sunken centres.

. Confirmation of Listeria spp. Suspect colonies from selective agars are streaked onto plates of tryptone soya yeast extract agar (TSYEA) and incubated at 37C for 18 to 24 h or until growth is sufficient. Select a typical (convex, colourless and opaque with an entire edge) isolated colony and carry out confirmatory tests (catalase, gram stain, umbrella or tumbling motility, -haemolysis, utilisation of rhamnose and xylose and CAMP test). MICRO-ID® Listeria or API®-Listeria and β-lysin CAMP factor discs (Remel #21-120, or equivalent) can be used. Strains that are considered to be Listeria monocytogenes must to be sent to a reference laboratory for further classification.

1 Agar Listeria according to Ottaviani and Agosti (ALOA), is a commercially available Listeria selective medium. 2 Any other solid selective media at the choice of the laboratory as long as it is complementary to ALOA Issue 2015 10 27 | Approved Methods Manual Export Standards Branch | Exports Division Page 1 of 2 Department of Agriculture and Water Resources Listeria Detection - AS 5013.24.1 - 2009

CHECKLIST

Primary Is the initial suspension prepared in half Fraser enrichment broth?

Is the primary enrichment at 30C for 24 ± 3 h? Are primary enrichment cultures (regardless of colour) inoculated into secondary enrichment medium? Is a positive control run with each batch of samples analysed? Are reference cultures inoculated into primary enrichment broth at a level of 10 to 100 cells?

Secondary Is secondary enrichment in Fraser broth? enrichment Is secondary enrichment carried out at 37C for 48 ± 3 h? Plating out and Are primary and secondary enrichment cultures identification (regardless of colour) streaked onto selective agars?

Is ALOA incubated at 37C for 24 ± 3 h? What is the second agar of choice and what are the incubation conditions? Are selective agars re-incubated for a further 18 to 24 h if growth is slight or no colonies are observed after 24 h?

Are descriptions of typical colony morphologies provided in the laboratories methods manual? Confirmation Is confirmation carried out on purified isolates from selective agars?

Are the following bio-chemical tests performed?

- Catalase ()

- Gram stain ()

- -Haemolysis ()

- Rhamnose ()

- Xylose (--)

- CAMP test Staph () Rhodo (--)

Issue 2015 10 27 | Approved Methods Manual Export Standards Branch | Exports Division Page 2 of 2 Department of Agriculture and Water Resources