2.2. Isolation of Listeria spp.
The isolation of Listeria spp. from brewer's grainswas performed
according to the double enrichment method (non-selective
enrichment and selective enrichment) followed by plate isolation
procedures described in the Bacteriological Analytical Manual of
the USA-FDA (Hitchins, 1998), with modifications in the media and
selective agents used.
Twenty five grams of each sample were homogenized in 225 mL
of tryptic soy broth (TSB) and incubated at 37 C for 24 h (nonselective
enrichment). Aliquots of 1 mL of primary enrichment
were transferred to 10 mL of TSB supplemented with tripaflavine
and ceftazidime and incubated at 4 C for 24 h and then at 37 C for
24 h (selective enrichment). Subsequently, aliquots of 0.1 mL of
secondary enrichments were plated in duplicate in Palcam agar
(Merck®, Germany) supplemented with selective agents. Typical
colonies selected by Gram staining were subcultured in tryptic soy
agar (TSA) for further biochemical characterization.