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
Department of Agriculture and Water Resources
Page 1 of 2
Listeria Detection - AS 5013.24.1 - 2009
CHECKLIST
Primary
enrichment
Is the initial suspension prepared in half Fraser
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
enrichment
Is secondary enrichment in Fraser broth?
Is secondary enrichment carried out at 37C for 48 ±
3 h?
Plating out and
identification
Are primary and secondary enrichment cultures
(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
Department of Agriculture and Water Resources
Page 2 of 2