listeria
TRANSCRIPT
Detection of Listeria monocytogenes Enrichment, Isolation and
Identification
Mm
more tha
n 100 ye
ars
Culture
Media M
erck
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.
2
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
3
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
5
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 _
6
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)
7
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
8
For further information: E. Merck • 64271 Darmstadt • Germany
Tel. ++496151/72 70 62 • Fax ++496151/72 77 14
Isolation media
Determination of b hemolysis