Chromocult Listeria Agar (Base) acc. OTTAVIANI 和 A

Technical Data Sheet

Chromocult

®

Listeria Agar (Base) acc. OTTAVIANI and AGOSTI

acc. ISO 11290

Ordering number: 1.00427.0500

For the isolation and differentiation of Listeria monocytogenes and other Listeria spp.

from food and animal feed, environmental samples in the area of food production and

food handling and other materials.

This culture medium complies with the specifications given by EN ISO 11290, FDA-BAM

and APHA.

Mode of Action

This medium contains the chromogenic compound 5-bromo-4- chloro-3-indolyl-β-D-

glucopyranoside, a substrate for the detection of β-glucosidase. This enzyme is common in

all Listeria, these appear on the medium as blue-green colored colonies.

Differentiation of Listeria monocytogenes from other Listeria spp. is achieved through the

production of a phosphatidylinositol-specific phospholipase C (PI-PLC). Listeria

monocytogenes hydrolyses the specific purified substrate added to the medium producing

an opaque halo around the colonies.

Most Listeria ivanovii also produce an opaque halo around the colonies after 48 h

incubation.

This medium contains a basis which ensures good growth for a broad range of bacteria. The

addition of inhibitors results in a marked reduction in the growth of the majority of

concomitant gram-positive and gram-negative pathogens, as well as of yeasts and fungi.

Selectivity is obtained by the addition of lithium chloride, nalidixic acid and cycloheximide,

whilst agar is the solidifying agent.

1.00427.0500

Page 1 of 7

Typical Composition

Specified by ISO 11290

Enzymatic Digest of Animal Tissues 18 g/l

Enzymatic Digest of Casein

Yeast Extract

Sodium Pyruvate

Glucose

Magnesium Glycerophosphate

MgSO

4

, anhydrous

NaCl

LiCl

Na

2

HPO

4

, anhydrous

5-Bromo-4-Chloro-3-Indolyl-

ß-D-Glucopyranoside

Agar

Nalidixic Acid Sodium Salt

Ceftazidime

Polymyxin B Sulfate

Amphotericin B

L-α-Phosphatidylinositol

Water

pH at 25 °C

6 g/l

10 g/l

2 g/l

2 g/l

1 g/l

0.5 g/l

5 g/l

10 g/l

2.5 g/l

Chromocult

®

Listeria Agar acc. OTTAVIANI

and AGOSTI acc. ISO 11290

Enzymatic Digest of Animal Tissues 18 g/l

Enzymatic Digest of Casein

Yeast Extract

Sodium Pyruvate

Glucose

Magnesium Glycerophosphate

MgSO

4

, anhydrous

NaCl

LiCl

6 g/l

10 g/l

2 g/l

2 g/l

1 g/l

0.5 g/l

5 g/l

10 g/l

2.5 g/l

0.05 g/l

13 g/l

0.02 g/l

0.02 g/l

76700 IU

0.01 g/l

2 g/l

n/a

7.2 ± 0.2

Na

2

HPO

4

, anhydrous

5-Bromo-4-Chloro-3-Indolyl-

0.05 g/l

ß-D-Glucopyranoside

12-18 g/lAgar-Agar*

0.02 g/l Nalidixic Acid Sodium Salt

0.02 g/l Ceftazidime

76700 IU Polymyxin B Sulfate

0.01 g/l Amphotericin B

2 g/l

L-α-Phosphatidylinositol

1000 ml/l Water

7.2 ± 0.2 pH at 25 °C

Supplements added after heating

* Agar-Agar is equivalent to other different terms of agar.

1.00427.0500

Page 2 of 7

Preparation

Dissolve 35 g of base medium in a total of 476 ml of purified water. For this, partially dissolve

the medium with 300 ml of water while swirling. Subsequently add the remaining 176 ml,

mix thoroughly, dissolve in boiling water, and agitate frequently until completely dissolved.

Then cool the medium in a water bath to 47-50 °C.

Add the contents of one vial of Selective Supplement (article number 1.00432.0010)

and distribute homogeneously.

Subsequently mix in the contents of one vial of Enrichment Supplement (article number

1.00439.0010) that has been pre-heated to 47-50 °C.

The antibiotic solution is sensitive to heat. Directly after adding the antibiotic and

enrichment solution pour plates up to a volume of 15-20 ml in dishes with a diameter of 90

mm.

The prepared medium is slightly opalescent to opalescent and yellowish.

There should be no visible moisture on the plates before use. When moisture is present,

the plates should be dried for the minimum time required to remove visible moisture,

following the procedure as described by EN ISO 11133.

Experimental Procedure and Evaluation

Depend on the purpose for which the medium is used.

Following the procedure given by EN ISO 11290-1, inoculate the surface of the medium from

the primary and secondary selective enriched cultures so that well-isolated colonies will be

obtained.

Following the procedure given by EN ISO 11290-2, inoculate the surface of the medium

direct with the initial suspension of the sample so that well-isolated colonies will be

obtained.

Incubate the inoculated plates inverted under aerobic conditions, e.g. acc. to EN ISO

11290 at 36-38 °C for 21-27 h, and if necessary after a further 21-27 h.

Examine the plates after the incubation for 21-27 h (and for an additional 21-27 h if the

growth is weak or if no colonies is observed after 24 h incubation), examine the plates

for the presence of colonies presumed to be Listeria spp.

Consider as L. monocytogenes the green-blue colonies surrounded by an opaque halo

(typical colonies). If growth is slight, or if no colony is observed, or if no typical colony is

present after 21-27 h incubation, re-incubate the plates for further 21-27 h and examine

again.

This presumptive evidence must be confirmed by carrying out the usual tests, e.g. those

described by EN ISO 11290.

Some strains of L. monocytogenes show a very weak halo (even no halo) in cases of stress,

in particular acid stress. Some rare L. monocytogenes are characterized by a slow PI-PLC

(phosphatidyl inositol phospholipase C) activity. Such bacteria are detected when the total

duration of incubation is more than, for example, 4 days; some of these strains could be

pathogenic (Leclercq, 2004).

No L. monocytogenes strains have been described as PI-PLC negative.

1.00427.0500

Page 3 of 7

Storage

Store at +15 °C to +25 °C, dry and tightly closed. Do not use clumped or discolored

medium. Protect from UV light (including sun light). For in vitro use only.

According Corry et al. (2012 self-prepared plates can be stored at +2 °C to +8 °C in the

dark and protected against evaporation at least four weeks

Quality Control

Function Control strains

Incubation

Reference

medium

Method

of control

Expected

results

Recovery

≥50 %, blue

green colonies

with opaque

halo

Listeria

monocytogenes

Productivity

1/2a ATCC

®

35152

Listeria

monocytogenes

4b ATCC

®

13932

Escherichia coli

ATCC

®

8739

Escherichia coli

ATCC

®

25922

Selectivity

Enterococcus

faecalis

ATCC

®

19433

Enterococcus

faecalis

ATCC

®

29212

Listeria

innocua ATCC

®

33090

40-48 h at

36-38 °C

Tryptic Soy

Quantitative

Agar (TSA)

40-48 h at

36-38 °C

- Qualitative Total inhibition

Specificity

40-48 h at

36-38 °C

-

No recovery limit

specified, blue

Qualitative

green colonies

without opaque

halo

Please refer to the actual batch related Certificate of Analysis.

The performance test is in accordance with the current version of EN ISO 11133. A recovery

rate of 50 % is equivalent to a productivity value of 0.5.

Listeria

monocytogenes

ATCC

®

13932

Listeria

innocua

®

ATCC 33090

1.00427.0500

Page 4 of 7

Literature

APHA (2015): Compendium of Methods for the Microbiological Examination of Foods. 5

th

ed.

American Public Health Association, Washington, D.C.

Bauwens, L., Vercammen, F. and Hensens, A. (2003): Detection of pathogenic Listeria

spp. in zoo animal faeces: use of immunomagnetic separation and a chromogenic isolation

medium. Vet. Microbiol. 91: 115 - 123.

Beumer, R.R. and Curtis, G.D.W. (2012): Culture media and Methods for the isolation

of Listeria monocytogenes. In: Handbook of Culture Media for Food and Water Microbiology.

(Corry, J.E.L., Curtis, G.D.W. and Baird, R.M. eds). pp. 115-129. Royal Society of

Chemistry, Cambridge, UK.

Corry, J.E.L., Curtis, G.D.W. and Baird, R.M. (2012): Handbook of Culture Media for Food

and Water Microbiology, pp. 658-662. Royal Society of Chemistry, Cambridge, UK.

FDA-BAM (2013): Chapter No. 10: Detection and Enumeration of Listeria monocytogenes

in Foods. U.S. Food and Drug Administration - Bacteriological Analytical Manual.

ISO International Standardisation Organisation. Microbiology of food and animal feeding

stuffs -- Horizontal method for the detection and enumeration of Listeria monocytogenes

- Part 1: Detection method -- Amendment 1: Modification of the isolation media and the

haemolysis test, and inclusion of precision data. EN ISO 11290-1:1998 + Amd 1:2004.

ISO International Standardisation Organisation. Microbiology of food and animal feeding

stuffs -- Horizontal method for the detection and enumeration of Listeria monocytogenes -

Part 2: Enumeration method - Amendment 1: Modification of the enumeration medium. EN

ISO 11290-2:1998 + Amd 1:2004.

ISO International Standardisation Organisation. Microbiology of food, animal feed and

water - Preparation, production, storage and performance testing of culture media. EN ISO

11133:2014.

Leclercq, A. (2004): Colonial atypical morphology and low recoveries of Listeria

monocytogenes strains on Oxford, PALCAM, Rapid' and ALOA solid media. J.

Microbiol. Methods. 57: 251-258.

Notermans, S.H.W., Dufrenne, J., Leimeister-Wachter, M., Domann, E. and Chakrabony,

T. (1991): Phosphatidylinositol-specific phospholipase C activity as a marker to distinguish

between pathogenic and non-pathogenic Listeria species. Appl. Environ. Microbiol. 57: 2666

- 2670.

Ottaviani, E.; Ottaviani, M. and Agosti, M. (1997): Differential agar medium for Listeria

monocytogenes. Ind. Aliment. 36: 888.

Vlaemynck, G., Lafarge, V. and Scotter, S. (2000): Improvement of the detection of

Listeria by the application of ALOA, a diagnostic, chromogenic isolation medium. J. Appl.

Microbiol. 88: 430 - 441.

1.00427.0500

Page 5 of 7

Ordering Information

Product

Chromocult

®

Listeria Agar (Base) acc.

OTTAVIANI and AGOSTI acc. ISO 11290

Chromocult

®

Listeria Agar

Enrichment Supplement

Chromocult

®

Listeria Agar

Selective Supplement

ReadyPlate™ CHROM Listeria Agar

ISO 11290

Cat. No.

1.00427.0500

1.00439.0010

1.00432.0010

1.46186.0020

Pack size

500 g

10 x1 vial

10 x1 vial

20 x 90 mm

500 g

60 bags

2000 ml bag

500 g

10 x 1 vial

10 x1 vial

20 x 10 ml

500 g

10 x 1 vial

20 pcs

500 g

10 x 1 vial

20 pcs

500 g

25 tests

25 tests

20 x 9 ml

Other pack

sizes available

100 x 90 mm

GranuCult™ Half FRASER (Demi FRASER) Broth

1.00025.0500

(Base) with Antibiotics acc. ISO 11290

Readybag

®

Half FRASER (Demi

FRASER) Broth with Supplements acc.

ISO 11290, 12.5 g, irradiated

GranuCult™ FRASER Broth (Base) acc.

ISO 11290

FRASER Listeria Selective Supplement

FRASER Listeria Ammonium Iron

(III) Supplement

ReadyTube™ 10 Fraser ISO 11290

Oxford-Listeria-Selective Agar (Base)

Oxford-Listeria-Selective Supplement

Oxford Listeria Selective Agar

PALCAM Listeria-Selective Agar (Base)

acc. to VAN NETTEN et al.

PALCAM Listeria Selective-Supplement

acc to van Netten et al

PALCAM Listeria Selective Agar

L-PALCAM-Listeria Selective Enrichment

Broth (Base) acc to van Netten et al.

Singlepath

®

Listeria

Singlepath

®

L’ mono

ReadyTube™ 9 BPW ISO 6579,

6887, 21528

1.02449.0060 35 x 62 g bags

6 x 225 ml

ReadyTube™ 2000 Half Fraser ISO 11290

1.46646.0001

1.10398.0500

1.00093.0010

1.00092.0010

1.46208.0020

1.07004.0500

1.07006.0010

1.46328.0020

1.11755.0500

1.12122.0010

1.46329.0020

1.10823.0500

1.04142.0001

1.04148.0001

1.46142.0020

100 x 10 ml

100 x 9 ml,

6 x 225 ml,

6 x 1000 ml,

1 x 2000 ml

1.00427.0500

Page 6 of 7

Merck KGaA

Frankfurter Strasse 250 64293

Darmstadt, Germany Fax: +49

(0) 61 51 / 72-60 80

Find contact information for your

country at:

/offices

For Technical Service, please visit:

/techservice

For more information, visit

/biomonitoring

Chromocult,

Darmstadt, Germany or its affiliates. Detailed information on trademarks is available

Merck, Millipore, and Sigma-Aldrich are trademarks of Merck KGaA,

via publicly accessible resources.

© 2019 Merck KGaA, Darmstadt, Germany and/or its affiliates. All Rights Reserved.

1.00427.0500

Page 7 of 7