HiCrome™ L.mono Rapid Differential Agar Base

Intended Use

for the rapid identification and differentiation of Listeria monocytogenes from other Listeria species based on rhamnose fermentation and PIPLC activity.

Composition**

Final pH ( at 25°C) 7.4±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 35.14 gram in 470 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Aseptically add sterile contents of 1 vial of LP Enrichment Supplement 1 (FD214) and sterile rehydrated contents of 1 vial of CA Selective Supplement (FD181) . 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, birth of a child with neonatal listeriosis and meningitis or primary bacteremia in adults and juveniles. The pathogenicity of Listeria ivanovii for humans is uncertain (1). Since L.monocytogenes and L. innocua have similar biochemical properties, they cannot be differentiated on traditional media (PALCAM, Oxford). This medium is based on the specific chromogenic detection of ß-glucosidase activity, rhamnose fermentation and PIPLC activity. Listeria species hydrolyse the purified chromogenic substrate in the medium giving blue coloured colonies. Since ß-glucosidase activity is specific for Listeria species, other organisms cannot utilize the chromogenic substrate and therefore give white colonies. Differentiation between Listeria species is based on the property of rhamnose fermentation and PIPLC activity. The colonies of L.monocytogenes appear bluish green with a yellow halo (rhamnose positive) while the colonies of L.ivanovii appear bluish green without a yellow halo (Rhamnose negative) (2,3). The differentiation of L. monocytogenes and L.innocua is based on PIPLC phosphatidylinositol-specific phospholipase C activity. Phospholipase C enzyme hydrolyses the purified substrate (FD214) added to the medium resulting in an opaque halo around Listeria monocytogenes colonies. L.ivanovii also demonstrates PIPLC activity however since it does not ferment rhamnose it can be easily distinguished from L.monocytogenes(1,4).

Peptone special, tryptone and soya peptone provide nitrogenous, carbonaceous substances, long chain amino acids, vitamin B complex and other essential growth nutrients. Rhamnose is the fermentable carbohydrate with phenol red as an indicator. Sodium chloride maintains the osmotic equilibrium. The added lithium chloride and CA Selective Supplement (FD181) inhibit growth of most gram-positive bacteria, gram-negative bacteria, yeasts and moulds. Phospholipase C enzyme hydrolyses the purified substrate (FD214) added to the medium resulting in an opaque halo around Listeria monocytogenes colonies demonstrating PIPLC activity.

Type of specimen

Food samples

Specimen Collection and Handling

For food samples, follow appropriate techniques for sample collection and processing as per guidelines (5,6). 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 organisms.
3. Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the COA based on the user’s unique requirement.
4. Further biochemical tests must be carried out for confirmation.

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 temperature.

Quality Control

Appearance Light yellow to pink homogeneous free flowing powder

Gelling Firm, comparable with 1.5% Agar gel.

Colour and Clarity of prepared medium Red coloured, opalescent gel forms in Petri plates

Reaction Reaction of 7.03% w/v aqueous solution at 25°C. pH : 7.4±0.2

pH 7.20-7.60

Cultural Response Cultural characteristics observed w/added CA Selective Supplement (FD181) and LP Enrichment Supplement 1(FD214), after an incubation at 35-37°C for 24-48 hours.

Key : (*) Corresponding WDCM numbers and **Formerly known as Bacillus subtilis subsp. spizizenii

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 (7,8).

Reference

1. Ottaviani F., Ottaviani M., and Agosti M. (1997 b), Quimper Froid Symposium Proceedings p. 6, A.D.R.I.A.Quimper, France, 16-18 June 1997.
2. Mengaud J., Braun-Breton C. and Cossart P., (1991), Molecular Microbiology, 5(2): 367-372.P
3. Notermans S.H. and Dufrenne J., (1991), Applied and Environmental Microbiology, 57(09): 2666-70.
4. Ottaviani F., Ottaviani M., and Agosti M. (1997 a), Industrie Alimentari 36, 1-3.
5. Salfinger Y., and Tortorello M.L. Fifth (Ed.), 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
6. Schlech WF, Lavigne PM, Bortolussi RA, et al.(January 1983). "Epidemic listeriosis-evidence for transmission by food".N. Engl. J.Med. 308(4): 203–6. doi:10.1056.
7. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
8. 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.