Automated BAX System for Detection of Listeria Monocytogenes in Foods AOAC 2003.12
Automated BAX System for Detection of Listeriamonocytogenes in Foods – AOAC 2003.12
SCOPE
This method as described here is applicable to:
Processed meat and poultry products and environmental samples.
PRINCIPLES
This method uses an automated commercial PCR screening procedure. All samples identified as potentially positive for Listeriamonocytogenes using this test must be confirmed using AS 5013.24.1 or MLG 8.The detection of Listeriamonocytogenes is broken down into four stages:
Pre-enrichment in non-selective liquid medium
A 25 g ± 1 g portion of sample is used for analysis of processed meat and poultry products. Primary enrichment is carried out in 225 ± 5 ml of modified University of Vermont broth (UVM) which is incubated at 30 ± 1C for 22-24 h. Environmental samples can be enriched in 60-100 ml UVM for sponges or in 10 ml if using small swabs.
Secondary enrichment and direct plating
Primary enrichment culture (0.1 ± 0.02 ml) is transferred to 10 ± 0.5 ml of MOPS-BLEB [1] broth. Secondary enrichment is carried out at 35 ± 1C for 18-24 h.
BAX system for screening Listeria
Listeriamonocytogenes is screened in the secondary enrichment broth following the manufacturer’s recommended protocol.
Confirmation
In all cases of BAX-positive, BAX-indeterminate or a BAX-signal-error the sample must be confirmed using AS 5013.24.1 (starting at the appropriate stage of analysis ie plating out and identification). The MOPS-BLEB cultures are streaked onto Oxford agar and PALCAM and incubated at 35 ± 1C [2] for 24 h and then for a further 18 to 24 h if growth is slight or if no colonies are observed after 24 h.
Typical Listeria colonies on Oxford agar are small (1 mm) greyish and surrounded by a black halos. After longer incubation colonies are larger and darker, with possibly a greenish colour, black halos and sunken centres. On PALCAM, Listeria 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. Suspect colonies are streaked onto plates of tryptone soya yeast extract agar (TSYEA) and incubated at 35 ± 1C for 18 to 24 h or until growth is sufficient. Typical colonies are selected (convex, colourless and opaque with an entire edge) and confirmed as Listeriamonocytogenes (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 Listeriamonocytogenes may be sent to a reference laboratory for further classification.
Issue 2015 10 27 | Approved Methods Manual
Export Standards Branch | Exports DivisionPage 1 of 2
Department of Agriculture and Water Resources
Listeria Detection - BAX – AOAC 2003.12
CHECKLIST
Primary enrichment / Is an appropriate initial suspension prepared in UVM broth?Is UVM incubated at 30 ± 2C for 22 ± 2 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 MOPS-BLEB?
Is secondary enrichment carried out at 35 ± 2C for 18-24 h?
BAX screening / Are positive and negative control run with each batch of samples?
Are the manufacturers instructions reproduced in the laboratory manual and followed without modification?
Plating out and identification / Are Oxford and PALCAM plates incubated at either 35 ± 1C for 24 h?
Are plates incubated for a further 18-24 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?
Is confirmation carried out for all BAX-positive, BAX-indeterminate or BAX-signal-error samples?
Confirmation / Is Listeria confirmed using AS 5013.24.1, MLG 8 or FDA BAM Chapter 10?
(if applicable) / If an external laboratory is used is it department approved?
Issue 2015 10 27 | Approved Methods Manual
Export Standards Branch | Exports DivisionPage 1 of 2
Department of Agriculture and Water Resources
[1]Listeria enrichment broth (BBL) to which is added 3-[N-Morpholino]propane sulfonic acid (6.7 g/L) & its sodium salt (10.5 g/L).
[2] 37 ± 1 C can be used for incubation of agar plates depending on the availability of suitable incubators. Whichever temperature is selected it must be used for all Listeria monocytogenes determinations undertaken at the laboratory.