Enterococcus Confirmatory Agar

The presence of intestinal enterococci, also known as fecal streptococci, is an indicator for fecal contamination, especially when the contamination occurred a long time ago and the less resistant coliform bacteria, including Escherichia coli, may already be dead at the time of analysis.

Casein peptone provides nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group, essential for bacterial growth. Dextrose is the fermentable carbohydrate providing carbon and energy. Sodium azide inhibits Gram-negative bacteria and methylene blue is an inhibitor of some Gram-positive bacteria. Methylene blue is also the pH indicator. Bacteriological agar is the solidifying agent.

To the prepared test tubes, in order to cover half of the slanted surface, aseptically add a volume of either Enterococcus Selective Broth (LC-1204) or Enterococcus Confirmatory Broth (same formulation as this medium but without the agar). Using growth from KAA Presumptive Broth (LC-1209), inoculate both the surface and the broth in the Confirmatory Agar/Broth mixture tube.

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 30.4 grams of the medium in one liter of distilled water. Dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into tubes and sterilize in autoclave at 121 °C for 15 minutes. Allow to cool in a slanted position in order to obtain butts of 1.5-2.0 cm depth.

Instructions for use

Incubate at 35 ± 2 °C for 18 hours and examine to detect the presence of small pinpoint colonies. Perform a Gram stain and observe under a microscope looking for long chains of ovoid cells. Immediately perform a catalase test by adding 5 mL of H₂O₂ to the tube in study. If there is no generation of gases (negative test), this constitutes the confirmation of enterococci in the sample.

• Winter and Sandholzer U.S. Dept. Interior Fishery, Leaflet 201 Part II, Nov. 1946.
• Ewing W.H. 1986. Edwards and Ewing's identification of Enterobacteriaceae 4th Edition.