KANAMYCIN AESCULIN AZIDE AGAR BASE

Code: CM0591

a selective medium when used with Kanamycin Supplement (SR0092) for the isolation of enterococci in foodstuffs

10.* Adjusted as required to meet performance standards

Directions
Suspend 21.3g in 500ml of distilled water. Add one vial of Kanamycin Supplement (SR0092) reconstituted as directed. Bring to the boil. Sterilise by autoclaving at 121°C for 15 minutes.

KANAMYCIN SULPHATE SUPPLEMENT

Code: SR0092

Directions
Add 2ml of sterile distilled water to one vial and mix gently to dissolve completely. Add the contents to 500ml of Kanamycin Aesculin Azide Agar Base.

Description
Kanamycin Aesculin Azide Agar was designed by Mossel et al.^1,2 to detect enterococci in foodstuffs. Round, white or grey colonies about 2mm in diameter, surrounded by black zones of at least 1cm diameter are considered to be enterococci (presumptive). Incubation is carried out aerobically at 35°C or 42°C ± 0.3°C for 18-24 hours. The higher incubation temperature increases the selectivity of the medium. This medium was used by Mossel et al.³ in the Dip Slide technique for bacteriological monitoring of foods. Kanamycin Aesculin Azide Agar has been used successfully for the isolation of glycopeptide-resistant enterococci from clinical specimens and foods^4,5.

Kanamycin Aesculin Azide Agar Base replaced Kanamycin Aesculin Azide Agar to follow the Oxoid Health & Safety rules that antibiotics should not be present in powdered culture media where they can be inhaled or contaminate surfaces. Kanamycin sulphate is added separately to 500ml of reconstituted agar from freeze-dried vials Kanamycin Sulphate Supplement (SR0092) containing the precise amount of antibiotic required.

The medium contains the selective inhibitory components kanamycin sulphate and sodium azide. It also contains an indicator system to detect the growth of aesculin-hydrolysing streptococci. These organisms produce black zones around the colonies from the formation of black iron phenolic compounds derived from aesculin-hydrolysis products and ferrous iron.

Technique
Inoculation method for samples: spread 0.1ml of sample dilutions over the whole of a pre-dried 9cm diameter plate.
The following procedure for testing foodstuffs is adapted from Mossel, Harrewijn and Elzebroek⁶.

1. Prepare tubes of sterilised Tryptone Water (CM0087) in 9ml volumes. Chill to 0-5°C by storing in a refrigerator for 18 hours prior to use.
2. Add 1g or 1ml of the thoroughly mixed food sample to a tube containing 9ml of pre-chilled diluent (10^-1 dilution). Shake well for 30 seconds. Sample 1ml of the contents, within 30 seconds after mixing, into a fresh tube of diluent. Continue the process using fresh sterile pipettes until a dilution is reached which will produce 100 colonies per 1ml. Store the decimal dilutions in the refrigerator and examine within 3 hours of their preparation.
3. Streak onto plates of Kanamycin Aesculin Azide Agar and incubate for 16-24 hours at 35°C ± 1°C. Consider the result positive for enterococci when colonies surrounded by black haloes are grown.
4. Confirmatory tests may be carried out, e.g. catalase test, utilisation of glucose, growth at 45°C ± 1°C, chain-forming Gram-positive cocci.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared media at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw/green coloured gel with blue tint

Quality control

* This organism is available as a Culti-Loop®

Precautions
Observe the hazard precautions regarding sodium azide when disposing of the medium.
There is no universal medium which will isolate all strains of enterococci⁷.
Unless a presumptive count is acceptable all isolates should have their identity confirmed with further tests.

References
1. Mossel D. A. A., Bijker P. G. H. and Eelderink I. (1978) Arch. Lebensmittel-hyg. 29. 121-127.
2. Mossel D. A. A., Bijker P. G. H., Eelderink I. and van Spreekens K. A. (1978) In: Streptococci. Eds. Skinner F.A. & Quesnel L.B. SAB Symposium. Series No.7. Academic Press. London.
3. Mossel D. A. A., Eelderink I., de Vor H. and Keizer E.D. (1976) Lab. Practice 25. 393-395.
4. Chadwick P.R., Brown D.F.J., Wilcox M.H. et al (1997) Clin. Microbiol. Inf. 3. 559-563.
5. Van den Braak N., Van Belkum A., Van Keulen M. et al. (1998) J. Clin. Microbiol. 36. 1927-1932.
6. Mossel D.A.A., Harrewijn G.A. and Elzebroek B.J.M. (1973) UNICEF Geneva.
7. Reuter G. (1985) Inter. J. Food Microbiol. 2. 103-114.