SUPPLEMENT TO METHOD MFLP-28

HEALTH PRODUCTS AND FOOD BRANCH
OTTAWA

Supplement to MFLP-28
January 2004
(PDF Version)

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1. APPLICATION

The following is offered as a supplement to the method MFLP-28, dated June 2003, and should be used with this method. The purpose of the supplement is to provide additional information on incubation time for both the Dairy and Other food categories. The media formulations are also included.

6. MATERIALS AND SPECIAL EQUIPMENT

Note: The Laboratory Supervisor must ensure that the analysis described in this method is carried out in accordance with the International Standard referred to as "ISO/IEC 17025:1999 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories".

Note: It is the responsibility of each laboratory to ensure that the temperatures of the incubators or water baths are maintained at the recommended temperatures. Where 35°C is recommended, the incubator may be 35 +/- 1°C. Similarly, lower temperatures of 30 or 25°C may be +/- 1°C. However, where higher temperatures are recommended, such as 43 or 45.5°C, it is imperative that the incubators or water baths be maintained within 0.5°C due to potential lethality of higher temperatures on the microorganism being isolated.

7. PROCEDURE

7.1. Collect and Enrich Samples

Sample Type	Primary enrichment	Secondary Enrichment
Processed Meat and Poultry	Weigh 25 g sample into sterile container. Homogenize with 225 mL UVM broth. Incubate 22-24 h at 30°C.	After 22-24 h transfer 0.1 mL of the enrichment to 9.9 mL MOPS-BLEB. Incubate 18-24 h at 35°C.
Raw Meat and Poultry Products	Weigh 25 g sample into sterile container. Homogenize with 225 mL Demi Fraser broth. Incubate 22-24 h at 30°C.	After 22-24 h transfer 0.1 mL of the enrichment to 9.9 mL MOPS-BLEB. Incubate 18-24 h at 35°C.
Dairy Products	Weigh 25 g sample into sterile container. Homogenize with 225 mL Complete Selective Enrichment broth. Incubate 22-24 h at 30°C.	After 22-24 h transfer 0.1 mL of the enrichment to 9.9 mL MOPS-BLEB. Incubate 18-24 h at 35°C.
Fruit, Vegetable and Seafood Products	Weigh 25 g sample into sterile container. Homogenize with 225 mL Enrichment broth. Incubate 4 h at 30°C. After 4 h, add selective agents acriflavin, nalidixic acid and cycloheximide. Incubate 22-26 h at 30°C.	After 22-26 h transfer 0.1 mL of the enrichment to 9.9 mL MOPS-BLEB. Incubate 18-24 h at 35°C.
Smoked Fish	Weigh 25 g sample into sterile container. Homogenize with 225 mL Universal Preenrichment broth. Incubate 22-26 h at 30°C.	After 22-26 h transfer 0.1 mL of the enrichment to 9.9 mL MOPS-BLEB. Incubate 18-24 h at 35°C.
Other Foods	Weigh 25 g sample into sterile container. Homogenize with 225 mL Enrichment broth. Incubate 4 h at 30°C. After 4 h, add selective agents acriflavin, nalidixic acid and cycloheximide. Incubate 22-26 h at 30°C.	After 22-26 h transfer 0.1 mL of the enrichment to 9.9 mL MOPS-BLEB. Incubate 18-24 h at 35°C.

9. PREPARATION OF MEDIA

9.1 Enrichment broth

Dispense into 225 mL and sterilize at 121°C for 15 min. Then add 2.5 mL 10% (w/v) filter-sterilized sodium pyruvate. Prepare acriflavin and nalidixic acid supplements as 0.5% (w/v) stock solutions in distilled water. Prepare cycloheximide supplement as 1.0% (w/v) stock solution in 40% (v/v) solution of ethanol in water. Add stock solutions: 0.45 mL acriflavin, 1.8 mL nalidixic acid and 1.15 mL cycloheximide to 225 mL enrichment broth. Final pH should be 7.3 +/- 0.1.

9.2 Complete Selective Enrichment broth

Adjust pH to 7.3 +/- 0.1. Dispense 225 mL and sterilize by autoclaving. Prepare acriflavin and nalidixic supplements as 0.5% (w/v) stock solutions in distilled water. Prepare cycloheximide supplement as 1.0% (w/v) stock solution in 40% (v/v) solution of ethanol in water. Add stock solutions: 0.45 mL acriflavin, 1.8 mL nalidixic acid and 1.15 mL cycloheximide to 225 mL enrichment broth.

9.3 UVM broth

Dispense into 225 mL and sterilize at 121°C for 15 min.

9.4 Demi-Fraser broth.

Adjust pH to 7.2 +/- 0.2. Dispense into 225 mL and sterilize at 121°C for 15 min.

9.5 MOPS-Buffered Listeria Enrichment broth (MOPS-BLEB).

Autoclave for 15 min at 121°C. Prepare 0.5% (w/v) acriflavin and naladixic acid stock solutions in distilled water. Prepare cycloheximide as a 1 % stock in 40% (v/v) ethanol/distilled water. All stock solutions are filter-sterilized. Add 3 mL acriflavin, 8 mL naladixic acid and 5 mL cycloheximide stock solutions to 1 L Listeria enrichment broth.

9.6 Universal Preenrichment broth.

Heat ingredients with gentle agitation to dissolve, dispense and autoclave at 121°C for 15 min. The final pH should be 6.3+/- 0.2.