Kanamycin Esculin Azide Agar Base

Intended Use

Recommended for selective isolation and identification of group D Streptococci in foodstuff.

Composition

Final pH (at 25°C): 7.0±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 21.32 grams in 500 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 and aseptically add rehydrated contents of one vial of KS Selective Supplement (FD146). Mix well before pouring into sterile petri plates.

Principle And Interpretation

Kanamycin Esculin Azide media are formulated as per Mossel et al (1,2) to detect Enterococci in food stuffs. Mossel et al (3) used it for the dip slide technique for bacteriological monitoring of foods. Tryptone, yeast extract provides nitrogeneous and carbonaceous compounds, long chain amino acids, vitamins and other essential nutrients for Enterococci. Kanamycin sulphate and Sodium azide are the selective inhibitory components. Esculin and Ferric ammonium citrate together form indicator system to detect esculin - hydrolysing Streptococci and imparts black zones around the colonies. Mossel et at (4) adopted the following procedure as - 1gm or 1ml mixed food is added to prechilled diluent (Tryptone water M463) and decimal dilutions are prepared. The decimal dilution are inoculated in Kanamycin Esculin Azide Broth (M776) and incubated at 35°C for 16-24 hours.

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 (5,6,7). 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

• The media is intended for primary isolation, further confirmatory tests to be done for complete identification.

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 light brown coloured homogeneous free flowing powder

Gelling: Firm, comparable with 1.0% Agar gel.

Colour and Clarity of prepared medium: Medium amber coloured clear to slightly opalescent gel with purplish tinge forms in Petri plates.

Reaction: Reaction of 4.26% w/v aqueous solution at 25°C. pH : 7.0±0.2

pH: 6.80-7.20

Cultural Response: Cultural characteristics observed with added KS Selective Supplement (FD146), after an incubation at 35-37°C or 42°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 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 (8,9).

Reference

1. Mossel D.A.A., Bijker, P.G.H. and Eelderink I., 1978, Arch. Lebensmittel - Hyg., 29:121.
2. Mossel D.A.A., et al., 1978, In: Streptococci., Skinner F.A. and Quesnel L. B. (ed.), SAB Symposium, series No.7, Academic Press, London.
3. Mossel D.A.A., et al, 1976, Lab. Practice, 25:393.
4. Mossel D.A.A., Harrenwijn G.A. and Elzebroek B.J.M., 1973, UNICEF Geneva.
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. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C.
7. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.
8. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
9. 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.