Isolation and Identification of Listeria Monocytogenes from Red Meat, Poultry, Egg And

Isolation and identification of Listeria monocytogenes from red meat, poultry, egg and environmental samples - MLG 8.10

SCOPE

Meat and meat products, eggs, carcase swabs and environmental samples.

PRINCIPLES

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

§  Primary enrichment

A 25 g ± 1 g portion is used for analysis of meat and meat products. Primary enrichment is carried out in 225 ± 5 ml of modified University of Vermont broth (UVM) which is incubated at 30 ± 2 °C for 20 - 26 h. Environmental sponge samples are enriched in 225±5 ml of UVM. For composite sponge samples add 100 ± 2 mL of UVM per sponge up to 5 sponges. Incubate at 30 ± 2 °C for 20 - 26 h.

§  Secondary enrichment

Primary enrichment culture (0.1 ± 0.02 ml) is transferred to 10 ± 0.5 ml of Fraser broth (FB) or MOPS-BLEB[1] and incubated at 35 ± 2 °C for 26 ± 2 h or 18-24 h, respectively. If no darkening of FB is evident after 26 ± 2 h then incubate until the total incubation time is 48 ± 2 h.

§  Plating out and identification

Primary enrichment broth (UVM) is streaked onto selective solid agar, Modified Oxford agar (MOX), after 20 - 26 h incubation. Selective agar plates are incubated at 35 ± 2 °C for 26 ± 2 h and then for a further 26 ± 2 h if growth is slight or if no colonies are observed after 26 ± 2 h.

FB showing any darkening after 26 ± 2 h are streaked onto MOX and incubated as detailed above. If no darkening in FB incubate for a total of 48±2 h. Re-examine after 48 ± 2 h. If no darkening and no typical colonies are evident on MOX plates, the sample is considered negative. Broths showing any darkening after 48 ± 2h are streaked onto MOX as previously described.

Streak 0.1 ml of MOPS-BLEB after 18-24 h onto MOX and incubate plates at 35±2 ºC for 26 ± 2 h.

§  Confirmation of Listeria spp.

If no darkening of FB is evident and no suspect MOX colonies have been identified, the sample is considered negative for L. monocytogenes. All suspect colonies on MOX are streaked for onto horse blood overlay agar at 35 ± 2 °C for 22 ± 4 h. Alternately, a swipe of suspect growth representing at least 20 colonies may be used. Colonies showing b-haemolysis are confirmed as L. monocytogenes using a commercially available bio-chemical test strip. Colonies should also be tested for tumbling motility and using the CAMP test. MICRO-ID® Listeria or API®-Listeria or VITE® 2 Compact (or equivalent) and β-lysin CAMP factor discs (Remel #21-120, or equivalent) can be used. Strains that are considered to be Listeria monocytogenes or are suspect but do not give definitive results must be sent to a reference laboratory for further classification.

§  Genetic Identification Test

Ribosomal RNA based test may be performed as a confirmatory test for all biochemically positive strains prior to confirmatory procedure

Issue 2017 03 22 | Approved Methods Manual

Export Standards Branch | Exports Division Page 2 of 2

Department of Agriculture and Water Resources

Listeria Detection - MLG 8.10

CHECKLIST

Primary enrichment / Is an appropriate initial suspension prepared in UVM broth?
Is UVM incubated at 30 ± 2°C for 20 - 26 h?
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 full strength Fraser broth?
Is secondary enrichment carried out at 35 ± 2°C for 26 ± 2 h?
Is FB incubated for a maximum of 48 ± 2 h if no darkening is evident after 26 h?
Plating out and identification / Are primary enrichment cultures streaked onto MOX at 20 - 26 h?
Are MOX plates incubated at either 35 ± 2°C for 26 ± 2 h?
Are MOX plates incubated for a further 20 -26 h if growth is slight or no colonies are observed after 26 h?
Are descriptions of typical colony morphologies provided in the laboratories methods manual?
Confirmation / Does the laboratory manual define what constitutes a negative sample?
Are all suspect colonies on MOX streaked to HL agar?
Are the following bio-chemical tests performed as a minimum?
- b-Haemolysis (Æ)
- Catalase (Æ)
- Rhamnose (Æ)
- Xylose (--)
- CAMP test Staph (Æ) Rhodo (--)
- CAMP/Factor test

Issue 2017 03 22 | Approved Methods Manual

Export Standards Branch | Exports Division Page 2 of 2

Department of Agriculture and Water Resources

[1] MOPS-BLEB Morpholinepropanesulfonic acid-buffered Listeria enrichment broth. Listeria enrichment broth (BBL) to which is added 3-[N-Morpholino]propane sulfonic acid (6.7 g/L) and its sodium salt (10.5 g/L)