SUPPLEMENT TO THE METHOD MFHPB-30

1. APPLICATION

The following information is offered as a supplement to the method MFHPB-30, dated January 2001, and should be used with this method. The purpose of this supplement is to provide additional information (such as source(s) of materials, critical steps, confirmation steps, or helpful hints etc.).

5. MATERIALS AND SPECIAL EQUIPMENT

Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency.

Listeria broths and agars (base media and supplements are commercially available)

7) Chromogenic media (follow manufacturer's instructions for preparation and use)
Rapid L. mono plates (Bio Rad Laboratories)
ALOA plates (AES Laboratoire)

6. PROCEDURE

6.6 Isolation Procedure

6.6.5 Chromogenic agar - novel chromogenic and other isolation agars may be used, but in conjunction with the plating media above. Follow manufacturer's instructions for preparation and use. On Rapid L. mono plates, L. monocytogenes forms blue colonies while other Listeria species are yellow to white in colour. On ALOA plates, L. monocytogenes colonies are blue-green with an opaque halo, while other Listeria species are blue-green without the halo.

6.7 Identification Procedure - Confirmation

6.7.3 Motility:

Agar: Stab motility test medium from selective agars, TA or TSA-YE. (Do blood agar and carbohydrates concurrently (see 6.7.2)). Incubate for 48 h (up to 7 days) at room temperature. Observe daily. ONLY Listeria cells give typical umbrella growth pattern.

and/or

Wet mount: follow that described in MFHPB-30, or alternately (from MFLP-38):

Pick at least one typical colony from each selective agar, TA or TSA-YE and do a wet mount examination using 0.85% saline for the suspending medium and the oil immersion objective of a phase-contrast microscope. Listeria spp. appear as slim, short rods with slight rotating or tumbling motility. Always compare to a known Listeria culture. Cocci, large rods, or rods with rapid, swimming motility are not Listeria spp.