See ISO 11290-1:1996 Microbiology of food and animal feeding stuffs -- Horizontal method for the detection and enumeration of
Listeria monocytogenes -- Part 1: Detection method, and ISO 11290-2:1998 Microbiology of food and animal feeding stuffs --
Horizontal method for the detection and enumeration of Listeria monocytogenes -- Part 2: Enumeration method.
RESULTS & INTERPRETATION
Listeria colonies appear as grey-green with a black precipitate, following inoculation and incubation at 35°C for 24-48 hours under
aerobic or microaerophillic conditions. L. monocytogenes does not ferment mannitol, thus colonies color will not change from red to
Listeria monocytogenes ATCC 7644 - good growth, brown-black / grey-green colonies with black center and black halo;
Enterococcus faecalis ATCC 29212 - inhibited / poor growth (brown colonies with brown-green halo);
Staphylococcus aureus ATCC 25923 - inhibited / poor growth (yellow colonies with yellow halo).
Columbia Blood Agar Base 39.0 g, Yeast extract 3.0 g, Glucose (dextrose) 0.5 g,
Aesculin 0.8 g, Ferric ammonium citrate 0.5 g, Mannitol 10.0 g, Phenol red 0.08 g,
Lithium chloride 15.0 g, Distilled water ad 1000 ml, pH 7.2 ± 0.2.
SELECTIVE SUPPLEMENT (for 500ml base medium)
Polymyxin B 10.0mg, Acriflavine hydrochloride 5.0mg, Ceftazidime 20.0mg.
Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C, and aseptically add the
Lithium chloride is harmful. Avoid bodily contact and inhalation of vapours.
Variants of medium replace Columbia Blood Agar Base & yeast extract with:
Peptic digest of animal tissue 23.0 g, Starch 1.0 g, Sodium chloride 5.0 g.
Medium contains two indicator systems, ferric citrate (esculin hydrolysis) and phenol
red (mannitol fermentation).
L. monocytogenes hydrolyzes esculin to form esculetin and dextrose. Esculetin reacts
with ammonium ferric citrate and forms a brown-black complex seen as a black halo
around colonies. Gram-negative bacteria are completely inhibited. Other
Gram-positives are inhibited or grow poorly.
Polymyxin-Acriflavin-Lithium chloride-Ceftazidime-Aesculin-Mannitol (PALCAM) Agar was formulated by Van Netten et al for the the
isolation of Listeria monocytogenes and other Listeria from foods.
(c) Costin Stoica