Foreword

Introduction

Hauptteil

1Scope

2Normative references

3Terms and definitions

4Principle

4.1General

4.2Primary enrichment in a selective liquid enrichment medium with reduced concentration of selective agents (half-Fraser broth)

4.3Secondary enrichment with a selective liquid enrichment medium with full concentration of selective agents (Fraser broth)

4.4Plating out and identification

4.5Confirmation

5Culture media and reagents

6Equipment and consumables

7Sampling

8Preparation of test sample

9Procedure

9.1Test portion and initial suspension

9.2Primary enrichment

9.3Secondary enrichment

9.4Plating out and identification

9.4.1General

9.4.2Agar Listeria according to Ottaviani and Agosti

9.4.3Second selective medium

9.5Confirmation of Listeria monocytogenes or Listeria spp.

9.5.1Selection of colonies for confirmation

9.5.2Confirmation of L. monocytogenes

9.5.2.1General

9.5.2.2Catalase reaction (optional)

9.5.2.3Motility test (optional)

9.5.2.4Microscopic aspect (optional in the case of use of agar specific for pathogenic Listeria spp.)

9.5.2.5Haemolysis tests

9.5.2.5.1General

9.5.2.5.2Haemolysis on blood agar

9.5.2.5.3Haemolysis reaction using red blood corpuscles

9.5.2.6CAMP test (optional)

9.5.2.7Carbohydrate utilization

9.5.3Confirmation of Listeria spp.

9.5.3.1General

9.5.3.2Catalase reaction

9.5.3.3Motility test (optional)

9.5.3.4Microscopic aspect

9.5.3.5Voges – Proskauer (VP) reaction (optional)

9.6Interpretation of morphological and physiological properties and of the biochemical reactions

9.7Additional characterization of isolated strains (optional)

10Expression of results

11Performance characteristics of the method

11.1Method validation studies

11.2Sensitivity

11.3Specificity

11.4Level of detection (LOD)

12Test report

13Quality assurance

Annex ADiagram of procedure (normative)

Annex BComposition and preparation of culture media and reagents (normative)

B.1Selective primary enrichment medium: half-Fraser broth

B.1.1Base

B.1.1.1Composition

B.1.1.2Preparation

B.1.2Lithium chloride solution

B.1.2.1Composition

B.1.2.2Preparation

B.1.3Solution of sodium salt of nalidixic acid

B.1.3.1Composition

B.1.3.2Preparation

B.1.4Acriflavine hydrochloride solution

B.1.4.1Composition

B.1.4.2Preparation

B.1.5Ammonium iron(III) citrate solution

B.1.5.1Composition

B.1.5.2Preparation

B.1.6Complete medium

B.1.6.1Composition

B.1.6.2Preparation

B.2Selective secondary enrichment medium: Fraser broth

B.2.1Base

B.2.1.1Composition

B.2.1.2Preparation

B.2.2Acriflavine hydrochloride solution

B.2.3Ammonium iron(III) citrate solution

B.2.4Complete medium

B.3Agar Listeria according to Ottaviani and Agosti1617

B.3.1Base medium

B.3.1.1Composition

B.3.1.2Preparation

B.3.2Nalidixic acid solution

B.3.3Ceftazidime solution

B.3.4Polymyxin B solution

B.3.5Antibiotic supplement

B.3.5.1Cycloheximide solution

B.3.5.2Amphotericin B solution (as an alternative to cycloheximide solution)

B.3.6Supplement

B.3.7Complete medium

B.3.7.1Composition

B.3.7.2Preparation

B.3.7.3Preparation of agar plates

B.4Second selective solid plating-out medium

B.5Performance testing for the quality assurance of the culture media

B.6Hydrogen peroxide solution

B.7Motility agar

B.7.1Composition

B.7.2Preparation

B.8Blood agar

B.8.1Base

B.8.1.1Composition

B.8.1.2Preparation

B.8.2Defibrinated blood (sheep, calf or bovine)

B.8.3Complete medium

B.8.3.1Composition

B.8.3.2Preparation

B.9Phosphate-buffered saline (PBS)

B.9.1Composition

B.9.2Preparation

B.10Red blood corpuscle suspension

B.11CAMP (Christie, Atkins, Munch-Petersen) medium and test strains

B.11.1General

B.11.2Base

B.11.3Blood medium

B.11.4Complete medium

B.12Carbohydrate utilization broth (L-Rhamnose and D-Xylose)

B.12.1Base

B.12.1.1Composition

B.12.1.2Preparation

B.12.2Carbohydrate solutions

B.12.2.1Composition

B.12.2.2Preparation

B.12.3Complete medium

B.13Reagents for Voges-Proskauer (VP) Reaction

B.13.1VP medium

B.13.1.1Composition

B.13.1.2Preparation

B.13.2α-Naphthol, ethanolic solution

B.13.2.1Composition

B.13.2.2Preparation

B.13.3Potassium hydroxide solution

B.13.3.1Composition

B.13.3.2Preparation

B.14Tryptone soya yeast extract agar (TSYEA)

B.14.1Composition

B.14.2Preparation

B.14.2.1Preparation of agar plates

Annex CDistinction of Listeria spp. from other genera (informative)

Annex DReactions for the identification of Listeria species (informative)

Annex EListeria selective agars (informative)

Annex FResults of the interlaboratory studies for detection of Listeria monocytogenes (informative)

F.1Results of interlaboratory studies

F.2Values of LOD from literature

Bibliography