Enterococcus Differential Agar Base (TITG Agar Base)

Intended Use

Recommended for the differentiation of Enterococcus faecalis and Enterococcus faecium.

Composition**

Final pH ( at 25°C): 6.05 ±0.05

**Formula adjusted, standardized to suit performance parameters

# - Equivalent to Beef extract

Directions

Suspend 43 grams in 1000 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 rehydrated contents of one vial of TTC solution 1% (FD057). Mix well and pour into sterile Petri plates.

Principle And Interpretation

Enterococci were formerly classified as faecal streptococci. Enterococci serves as an indicator organism in monitoring food samples as it is cause of faecal contamination. Of the various species of Enterococci, E. faecalis and E. faecium are frequently found in humans. The presence of Enterococci in food samples has been studied. (3,7).

A variety of selective media have been recommended for the isolation of Enterococcus species (4). Enterococcus Differential Agar Base was designed for the selective isolation and differentiation between Enterococcus faecalis and Enterococcus faecium. The differentiation is based depending upon the reduction of tetrazolium. Enterococcus faecalis produces colonies with a deep red centre and a narrow white periphery, whereas Enterococcus faecium produces white or pale pink coloured colonies.

Proteose peptone and HM peptone B serves as a source of nitrogen, carbon, long chain amino acids and vitamins. Glucose serves as a source of carbohydrate. The medium incorporates thallium acetate as a selective inhibitory agent (2).

Type of specimen

Food and dairy samples.

Specimen Collection and Handling

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (1,8,9). 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. Some species may show poor growth due to variable nutritional requirements.

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: Cream to yellow homogeneous free flowing powder

Gelling: Firm, comparable with 1.4% Agar gel

Colour and Clarity of prepared medium: Light yellow coloured clear to slightly opalescent gel forms in Petri plates

Reaction: Reaction of 4.30% w/v aqueous solution at 25°C. pH : 6.05±0.05

pH: 6.00-6.10

Cultural Response

Cultural characteristics observed with added TTC Solution 1% (FD057) after an incubation at 35-37°C for 18-24 hours.

Key : (*) Corresponding WDCM numbers.

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°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 clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (5,6).

Reference

1. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C.
2. Barnes,E.M.(1956) Methods for the isolation of faecal streptococci (Lancefield group D) from bacon factories.J.Appl.Bacteriol.19,193-203.
3. Devriese, L.A., Pot, B., Van Damme, L., Kersters, K and Haesebrouk, F. (1995) Identification of Enterococcus species isolated from food of animal origin. Int. J. Food Microbiol. 26, 187-197.
4. Domig, K.J., Mayer, H.K. and Kneifel, W (2003a) Methods used for isolation, enumeration, characterization and identification of Enterococcus species.1. Media for isolation and enumeration. Int.J.Food Microbiol.88 147-164.
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.
7. Knudtson, L.M. and Hartman, P.A. (1993) Enterococci in pork processing. J.Food Prot. 56, 6-9.
8. 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.
9. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.