Detection of Listeria monocytogenes

Enrichment, Isolation and

Identification
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 Listeria monocytogenes:
Importance, occurrence and identification
Since 1985, after several deaths in the USA and
Switzerland due to the consumption of contami-
nated soft cheese products, Listeria bacteria have
become the subject of public attention.
The infections caused by L. monocytogenes range
from ailments similar to influenza, up to meningitis
and encephalitis. The pathogens can also cause
pus formation, abscesses or granulomas similar to
those found in tuberculosis in internal organs.
Afflications occur particularly in people with
weakened resistance such as small children, preg-
nant women, old people and patients treated with
immunosuppressive agents.
L. monocytogenes is widely spread in the environ-
ment and is found for example in the earth, in
water, sewage, silage, animal feed stuffs as well as
in man and animals.
Therefore its presence in animal or vegetable foods
is almost unavoidable.
Infections however are rare although Listeria is so
widely spread. We must presume though that in
some cases the infection is not recognised as the
symptoms are variable and partly unspecific. The
L. monocytogenes germ belongs to the group of
Single colonies of L. monocytogenes
Palcam Agar
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bacteria which are relativily simple to grow.
Generally the usual solid, liquid or semi-solid
culture media are adequate.
L. monocytogenes can grow within a wide tempe-
rature range (+ 3 °C to 45°C). The optimum
growth temperature lies between + 30 °C and
+ 37 °C.
No particular atmospheric conditions are neces-
sary. Reproduction is aerobic but can be improved
by a microaerophilic atmosphere. A completely
anaerobic atmosphere gives very poor growth re-
sults. The following culture media are described
and recommended for the isolation of L. monocyto-
genes.
The isolation of L. monocytogenes is still very diffi-
cult however because of the small number of these
bacteria which are usually present. Also, as a
small colony they are relatively quickly overgrown
by other bacteria.
All efforts therefore concentrate on suppressing
possible accompanying flora, then it remains very
difficult to culture and isolate Listeria particularly
from examination material from mixed infections.
Oxford Agar
 Diagram of Procedure acc. to IDF-FIL-*Method
Detection of Listeria monocytogenes
in Milk & Milk Products

Homogenize 25 g/ml of sample

in 225 ml LEB Broth
Incubation: 48 h at 30 °C

Streakout either/or on
PALCAM or OXFORD Agar

Incubation: 48 h at 30 °C

5 suspect colonies

TSA-YE Agar

Incubation: 24 h at 37 °C

Further examinations

*International Dairy Federation

Fédération Internationale de Laiterie 3
 Diagram of Procedure Diagram of Procedure
acc.to FDA-*Method acc.to USDA-*Method
Detection of Listeria monocytogenes Detection of Listeria monocytogenes
in food in meat/poultry products
Homogenize 25 g/ml of sample
Homogenize 25 g/ml of sample
in 225 ml UVM I Broth
in 225 ml LEB Broth
Incubation: 48 h at 30 °C

Subculture 0,1 ml in 10 ml UVM II Broth

Incubation: 24 h and 48 h at 30 °C after 24 h incubation time

after 24 h subculture after 48 h subculture Incubate: 24 h at 30 °C

Streakout on LPM Agar

Oxford Agar LPM Agar LPM Agar Oxford Agar
35 °C 30 °C 30 °C 35 °C

Incubation: 24 h at 48 °C Incubation: 24 h at 30 °C

5 suspect colonies 5 suspect colonies

TSA-YE Agar TSA-YE Agar

Incubation: 24 h at 48 h at 30 °C Incubation: 24 h at 35 °C

Further examinations Further examinations

*Food and Drug Administration *United States Department of Agriculture

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 Diagram of Procedure acc.to ISO/CD* draft 11290 and
Methode de Routine AFNOR* V08-055
Detection of Listeria monocytogenes

Homogenize 25 g/ml of sample

in 225 ml FRASER Broth (containing 1/2 concentration of inhibitors)
Incubation: 24 h at 30 °C

Subculture 0.1 ml in
10 ml full-strength
FRASER Broth

Incubate 24 to 48 h at 35 or 37 °C

Streakout either/or After 24 h streakout either/or After 48 h streakout either/or

on PALCAM or OXFORD Agar on PALCAM or OXFORD Agar on PALCAM or OXFORD Agar

Incubation: 24 h, if necessary up to 48 h at 30 or 37 °C

5 suspect colonies 5 suspect colonies 5 suspect colonies

TSA-YE Agar TSA-YE Agar TSA-YE Agar

Incubation: 24 h at 30 °C or 35 - 37 °C

Further examinations Further examinations Further examinations

*International Standard Organization/Committee Draft

Association Francaise de Normalisation 5
 Biochemical Reactions of Listeria Species
L. monocy- L. wels- L. denitri-
Species L. innocua L. ivanovii L. seeligeri L. grayi L. murrayi
togenes himeri ficans

Beta hemolysis + _ + + _ _ _ _

Catalase + + + + + + + +

Nitrate reduction _ _ _ _ _ + _ +

Urea hydrolysis _ _ _ _ _ _ _ _

Methyl red + + + + + + + +

Voges-Proskauer + + + + + _ + +

TSIA A/Aa A/A A/A A/A A/A A/A A/A A/A

SIM motility + + + + + + + +

Dextrose + + + + + + + +

Esculin + + + + + + + +

Maltose + + + + + + + +

Mannitol _ _ _ _ _ _ + +

Rhamnose + Vb _ _ V _ _ V

Xylose _ _ + + + + _ _

a Acid slant/acid butt / b Variable (Lovett, J. 1988)

Biochemical characteristics of the genus Listeria

Characteristic Reaction

Motility (at 20 - 25 °C) +

Oxygen requirement facultative

Growth at 35 °C +

Catalase activity +

Hydrogen sulphide production _ CAMP-Test

Acid from glucose +

Methyl red reaction +

Voges-Proskauer reaction +

Indole production _

Citrate utilisation _

Urease activity _
L. monocytogenes (Beta hemolysis)

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 Whatever you need, Merck offers the full range
of products for the detection of Listeria
Enrichment

Cat.No. Designation Package

1.11951.0500 Listeria Enrichment Broth (LEB) acc.to FDA/IDF-FIL 500 g

1.11781.0001 Listeria LEB Supplement acc.to IDF-FIL 16 vials

1.11883.0001 Listeria LEB Supplement acc.to FDA 16 vials

1.10823.0500 L-PALCAM Listeria Enrichment Broth (Base) 500 g

1.12122.0001 PALCAM Selective Supplement 16 vials

1.10824.0500 UVM Listeria Enrichment Broth (Base) 500 g

1.04039.0001 UVM II Supplement 1 vial

1.10398.0500 FRASER Listeria Enrichment Broth (Base) 500 g

1.10399.0001 FRASER Listeria Supplement 16 vials

1.10985.0500 Listeria Enrichment Broth (Base) acc.to FEINDT 500 g

L - Palcam Broth FRASER Broth

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 Isolation media

Cat.No. Designation Package

1.07004.0500 OXFORD Listeria Agar 500 g

1.07006.0001 OXFORD Listeria Selective Supplement 13 vials

1.11755.0500 PALCAM Listeria Agar acc.to van Netten et.al 500 g

1.12122.0001 PALCAM Listeria Selective Supplement 16 vials

1.10986.0500 Listeria Selective Agar acc.to FEINDT 500 g

Determination of b hemolysis

1.10886.0500 Blood Agar (Base) 500 g

References
Van Netten, P. et al.:
Liquid and solid selective differential media for the detection and enumeration of Listeria monocytogenes
International Journal of Food Microbiology, 8 (4), 299 - 316 (1989)
Seeliger,H. P. R., & Jones, D.:
In "Bergey`s Manual of Systematic Bacteriology", Vol. 2, 1235 - 1245 (1986)
Lovett, J.:
Isolation and enumeration of Listeria monocytogenes
Food Technology 42 (4), 172 - 175 (1988)
Curtis, G. D. W., et al.:
A selective medium for the isolation of Listeria monocytogenes
Letters in Applied Microbiology 1989, 8, 95 - 98
Hammer P., et al.:
Vergleich der Eignung von Oxford- und PALCAM-Medium zur Isolierung von Listeria monocytogenes aus
Weichkäse
Deutsche Milchwirtschaft 11/1990
Provisional International IDF Standard 143: 1990
Milk & Milk products
Detection of Listeria Monocytogenes

For further information:

E. Merck • 64271 Darmstadt • Germany
Tel. ++496151/72 70 62 • Fax ++496151/72 77 14
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