HiCrome™ L mono differential Agar Base

Intended use

Recommended for the selective and differential isolation, enumeration and identification of Listeria monocytogenes and Listeria species based on PCPLC activity.

Composition**

Final pH ( at 25°C): 7.2±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 33.60 gram in 480 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Aseptically add sterile contents of 1 vial of Lecithin solution (FD332) and sterile rehydrated contents of PANC Selective Supplement (FD333). Mix well and pour into sterile Petri plates.

Principle And Interpretation

Listeria monocytogenes is a gram-positive foodborne human pathogen responsible for serious infections in pregnant women that may ultimately result in abortion, stillbirth, and birth of a child with neonatal listeriosis and meningitis or primary bacteremia in adults and juveniles (1). The pathogenicity of Listeria ivanovii for humans is uncertain. Since L.monocytogenes and L.innocua have similar biochemical properties, they cannot be differentiated on traditional media (PALCAM, Oxford). HiCrome® L.mono Differential Agar Base is based on for the selective and differential isolation of Listeria species on the basis of utilization of chromogenic substrate and lecithinase activity [Phosphotidylcholine phospholipase C (PCPLC)] (2). PI-PLC and PC-PLC, the major virulence factors, are only produced by pathogenic L. monocytogenes and Listeria ivanovii (3). Peptone, tryptone, yeast extract and sodium pyruvate provide nitrogenous and carbonaceous compounds, long chain amino acids, vitamins and essential growth nutrients. Maltose is the fermentable carbohydrate. Sodium chloride maintains osmotic equilibrium. Phosphate buffers the medium. Lithium chloride and added selective supplement (FD333) inhibit accompanying microflora and allow the growth of Listeria species. Listeria species hydrolyse the chromogenic substrate and produces green coloured colonies. Lecithin solution (FD332) helps in detecting PCPLC activity. Differentiation of Listeria species is based on phosphatidylcholine phospholipase C (PCPLC) activity. L.monocytogenes and L.ivanovii exhibits PCPLC activity which is seen as opaque halo around the colony.

Type of specimen

Food samples

Specimen Collection and Handling

For food samples, follow appropriate techniques for sample collection and processing as per guidelines (4). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
2. Slight colour variation may be observed depending upon the utilization of the substrate by the organism.
3. Further confirmation has to be carried using sensitivity testing.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temmperature.

Quality Control

Appearance: Cream to yellow homogeneous free flowing powder

Gelling: Firm, comparable with 1.4% Agar gel

Colour and Clarity of prepared medium: Light amber coloured, opalescent gel forms in Petri plates

Reaction: Reaction of 6.72% w/v aqueous solution at 25°C. pH : 7.2±0.2

pH: 7.00-7.40

Cultural Response: Cultural characteristics observed with added sterile PANC Selective Supplement (FD333) and Lecithin solution (FD332) after an incubation at 35 - 37°C for 24 - 48 hours.

Key : (* )- Corresponding WDCM Numbers

Storage and Shelf Life

Store between 15-25°C in a tightly closed container and the prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques (5,6).

Reference

1. Painter J, Slutsker L. 2007. Listeriosis in humans, p 85–109. In Ryser ET, Marth EH (ed), Listeria, listeriosis, and food safety. Marcel Dekker, New York, NY.
2. Sang-Hyun Park, Pahn-Shick Chang, Sangryeol Ryu and Dong-Hyun Kang. Development of a Novel Selective and Differential Medium for the Isolation of Listeria monocytogenes. Applied and Environmental Microbiology 2014.
3. Mengaud J, Braun-Breton C, Cossart P 1991. Identification of phosphatidylinositol-specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor. Mol. Microbiol. 5:367–372. doi:10.1111/j.1365-2958.1991
4. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2001, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
5. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
6. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.