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 andSwitzerland due to the consumption of contami-nated soft cheese products, Listeria bacteria havebecome the subject of public attention.

The infections caused by L. monocytogenes rangefrom ailments similar to influenza, up to meningitisand encephalitis. The pathogens can also causepus formation, abscesses or granulomas similar tothose 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 withimmunosuppressive 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 asin man and animals.

Therefore its presence in animal or vegetable foodsis almost unavoidable.

Infections however are rare although Listeria is sowidely spread. We must presume though that insome cases the infection is not recognised as thesymptoms are variable and partly unspecific. TheL. monocytogenes germ belongs to the group of

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 improvedby a microaerophilic atmosphere. A completelyanaerobic atmosphere gives very poor growth re-sults. The following culture media are describedand 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 thesebacteria which are usually present. Also, as asmall colony they are relatively quickly overgrownby other bacteria.

All efforts therefore concentrate on suppressingpossible accompanying flora, then it remains verydifficult to culture and isolate Listeria particularlyfrom examination material from mixed infections.

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Palcam Agar Oxford Agar

Single colonies of L. monocytogenes

Diagram of Procedure acc. to IDF-FIL-*Method Detection of Listeria monocytogenes

in Milk & Milk Products

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Homogenize 25 g/ml of sample in 225 ml LEB Broth

Incubation: 48 h at 30 °C

Streakout either/or onPALCAM 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 FederationFédération Internationale de Laiterie

Diagram of Procedure acc.to FDA-*Method

Detection of Listeria monocytogenes in food

Diagram of Procedure acc.to USDA-*Method

Detection of Listeria monocytogenes in meat/poultry products

Incubation: 24 h and 48 h at 30 °C

Incubation: 24 h at 48 h at 30 °C

Further examinations

after 24 h subculture after 48 h subculture

Oxford Agar35 °C

LPM Agar30 °C

LPM Agar30 °C

Oxford Agar35 °C

Homogenize 25 g/ml of sample in 225 ml LEB Broth

5 suspect colonies

TSA-YE Agar

Incubation: 24 h at 48 °C

Homogenize 25 g/ml of sample in 225 ml UVM I Broth

Incubation: 48 h at 30 °C

Subculture 0,1 ml in 10 ml UVM II Brothafter 24 h incubation time

Incubate: 24 h at 30 °C

5 suspect colonies

TSA-YE Agar

Incubation: 24 h at 35 °C

Further examinations

Streakout on LPM Agar

Incubation: 24 h at 30 °C

4*Food and Drug Administration *United States Department of Agriculture

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

Detection of Listeria monocytogenes

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Subculture 0.1 ml in 10 ml full-strengthFRASER Broth

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

Further examinations

After 24 h streakout either/oron PALCAM or OXFORD Agar

Homogenize 25 g/ml of sample in 225 ml FRASER Broth (containing 1/2 concentration of inhibitors)

Incubation: 24 h at 30 °C

5 suspect colonies

TSA-YE Agar

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

Further examinations

TSA-YE Agar

Further examinations

TSA-YE Agar

Incubate 24 to 48 h at 35 or 37 °C

After 48 h streakout either/oron PALCAM or OXFORD Agar

Streakout either/oron PALCAM or OXFORD Agar

5 suspect colonies 5 suspect colonies

*International Standard Organization/Committee DraftAssociation Francaise de Normalisation

Biochemical Reactions of Listeria Species

Biochemical characteristics of the genus Listeria

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

Characteristic Reaction

Motility (at 20 - 25 °C) +

Oxygen requirement facultative

Growth at 35 °C +

Catalase activity +

Hydrogen sulphide production _

Acid from glucose +

Methyl red reaction +

Voges-Proskauer reaction +

Indole production _

Citrate utilisation _

Urease activity _

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SpeciesL. monocy-

L. innocua L. ivanovii L. seeligeriL. wels- L. denitri-

L. grayi L. murrayitogenes 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 _ _ + + + + _ _

CAMP-Test

L. monocytogenes (Beta hemolysis)

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

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

Enrichment

References

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

1.10886.0500 Blood Agar (Base) 500 g

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

WeichkäseDeutsche Milchwirtschaft 11/1990

Provisional International IDF Standard 143: 1990Milk & Milk products

Detection of Listeria Monocytogenes

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For further information: E. Merck • 64271 Darmstadt • Germany

Tel. ++496151/72 70 62 • Fax ++496151/72 77 14

Isolation media

Determination of b hemolysis