Listeria Agar Base Palcam ISO

Principles and uses
Listeria Agar Base Palcam, used with supplements, is a selective and differential medium for Listeria spp. It is recommended by ISO 11290 for the detection and enumeration of Listeria monocytogenes in food products and clinical samples, and can also be used for environmental samples.

It is used after a primary and secondary enrichment stage, using Listeria Enrichment Broth Base (Cat.1120). It allows the easy differential diagnosis of Listeria monocytogenes using a double-system indicator: Esculin/Iron and Mannitol/Phenol red. All Listeria species hydrolyze the esculin to esculetin, which reacts with iron ions producing a blackening of the medium.
Lithium chloride included in the medium, along with ceftazidime, polymyxin B sulfate and Acryflavine from the supplement, inhibit the growth of the non-Listeria accompanying bacteria present in foods, which can hydrolyze the esculin. Peptones and maize starch provide a rich nutrient base for growth. Yeast extract is the source of vitamins, particularly of the B-group. Glucose is the fermentable carbohydrate. Ferric ammonium citrate improves the growth of L. monocytogenes.
The Mannitol/Phenol red differentiation system is used to differentiate Listeria spp that do not ferment mannitol from other species that occasionally grow in the medium such as enterococci or staphylococci. Differentiation is achieved by the acid increase in the media, causing the phenol red indicator to change the color of the medium from red to yellow. Confirmation of Listeria is done by biochemical and serological identifications tests.

Preparation
Suspend 34,4 grams of the medium in 500 ml of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Sterilize in autoclave at 121 ºC for 15 minutes. Cool to 45-50 ºC and aseptically add one vial of Palcam Listeria Selective Supplement. Homogenize gently and dispense into Petri dishes.

Instructions for use
For clinical diagnosis, the type of sample is amniotic fluid.
- Inoculate on the surface making parallel striae with the handle or swab

- Incubate in aerobic conditions at 35±2 ºC for 24-48 hours.
- Reading and interpretation of the results.

For the detection and enumeration of Listeria monocytogenes and Listeria spp. according to ISO 11290:
Primary enrichment:
- Weigh 25 g (or 25 ml) of the sample and add 225 ml of Listeria 1/2 Fraser Broth. Homogenize and incubate at 30 ºC for 25±1 h.
Secondary enrichment:
- Inoculate 0,1 ml of the culture of the Listeria 1/2 Fraser Broth incubated (regardless of its color) in 10 ml of Listeria Fraser Broth .
Incubate at 37 ºC for 24±2 hours under aerobic conditions.
Plaque and identification:
- From the primary enrichment culture, the Listeria Agar surface is inoculated according to Ottaviani and Agosti , to obtain well separated colonies.
- From the secondary enrichment culture, the procedure is repeated, inoculate the surface of the Listeria Agar according to Ottaviani and Agosti, the Palcam Listeria Agar  and another medium such as the Oxford Agar.
- For Listeria Agar according to Ottaviani and Agosti incubate for a total of 48±2 h.
- For Agar Lisetria Palcam incubate at 35±2 ºC for 24-48 h.
- For Oxford agar incubate at 35±2 ºC for 24-48 h.

Confirmation:
- Select the presumptive colonies and carry out confirmatory tests for L. monocytogenes or Listeria spp.