FSIS Procedure for the use of the Listeria monocytogenes BAX Screening
Test - MLG 8A.05
SCOPE
This method is applicable to processed meat and poultry products and environmental
samples.
PRINCIPLES
This method uses a commercial PCR screening procedure. All samples identified as
potentially positive for Listeria monocytogenes using this test must be confirmed using AS
5013.24.1 or MLG 8.
The detection of Listeria monocytogenes is broken down into four stages:

Pre-enrichment in non-selective liquid medium
A 25 g ± 1 g portion 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 ± 2C for 20 - 26 h. Environmental swab samples
are enriched in 225± 5 ml of UVM and incubated at 30 ±2C for 20 - 26 h.

Secondary enrichment and Primary enrichment 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 ± 2C for 18-24 h. Primary
enrichment cultures are also streaked onto selective solid agar (MOX) after 20 – 26 h
incubation. Selective agar, Modified Oxford agar (MOX), is incubated at 35 ± 2C for 26
± 2 h.

BAX system for screening Listeria
Listeria monocytogenes is screened in the secondary enrichment broth following the
manufacturer’s recommended protocol2. The BAX Listeria monocytogenes test is an
automated method that uses polymerase chain reaction (PCR) technology for the
detection of Listeria monocytogenes in food and environmental samples. The system
identifies a specific DNA fragment unique to Listeria monocytogenes. The DNA is
combined with DNA polymerase, nucleotides, and primers. The mixture then undergoes
a series of timed heating and cooling cycles. Heating denatures the DNA, separating it
into single strands. As the mixture cools, the primers recognise and bind to the targeted
DNA sequences. The DNA polymerase then uses the nucleotides to extend the primers,
thus creating 2 copies of the targeted DNA fragment; repeating the cycle results in an
exponential increase in the number of target DNA fragments. A fluorescent dye then
binds with double-strand DNA and emits a fluorescent signal in response to light. After
amplification, the BAX System begins a detection phase in which the fluorescent signal
is measured.
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
The FSIS use DuPont Qualicon kits #17710609 held at 4 ± 2 C.
Issue 2015 10 27 | Approved Methods Manual
Export Standards Branch | Exports Division
Department of Agriculture and Water Resources
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Listeria Detection - FSIS BAX – MLG 8A.05

Confirmation
BAX-negative samples that have typical colonies on 26 ± 2h MOX plates or BAX-positive,
BAX-indeterminate or a BAX-signal-error the sample must be confirmed using AS
5013.24.1 or MLG 8 (starting at the appropriate stage of analysis i.e. plating out and
identification). The MOPS-BLEB culture is streaked onto MOX and incubate 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 h. Confirmation then proceeds as described in AS 5013.24.1 or MLG 8.

Confirmation of Listeria spp
Suspect colonies from selective agars are streaked onto plates of tryptone soya yeast
extract agar (TSYEA) and incubated at 35 ± 1C 3 Error! Bookmark not defined. 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/CAMP factor test). MICRO-ID® Listeria or API®-Listeria and β-lysin CAMP factor
discs (Remel #21-120, or equivalent) can be used or well established schemes
(Compendium of methods for the Microbiological Examination of Foods, Bacteriological
Analytical Manual). Strains that are considered to be Listeria monocytogenes may be
sent to a reference laboratory for further classification.
MLG 8
All suspect colonies on MOX are streaked onto horse blood overlay agar at 35 ± 2C for
22 ± 4 h. Colonies showing -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/CAMP factor test. 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.
AS 5013.24.1
Suspect colonies from MOX are streaked onto plates of tryptone soya yeast extract agar
(TSYEA) and incubated at 35 ± 1C 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 Listeria monocytogenes (catalase, gram stain, umbrella or tumbling
motility, -haemolysis, utilisation of rhamnose and xylose and CAMP/CAMP Factor test).
Strains that are considered to be Listeria monocytogenes may be sent to a reference
laboratory for further classification.
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.
3
Issue 2015 10 27 | Approved Methods Manual
Export Standards Branch | Exports Division
Department of Agriculture and Water Resources
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Listeria Detection - FSIS BAX – MLG 8A.05
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 MOPS-BLEB?
Is secondary enrichment carried out at 35 ± 2C
for 26 ± 2 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 primary enrichment cultures streaked onto
MOX?
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?
Are MOPS-BLEB cultures streaked onto MOX for
all BAX-positive, BAX-indeterminate or BAXsignal-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 Division
Department of Agriculture and Water Resources
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