KF STREPTOCOCCUS AGAR BASE

Code: CM0701

a selective medium for the isolation and enumeration of enterococci

Typical Formula*	gm/litre
Proteose peptone	10.0
Yeast extract	10.0
Sodium chloride	5.0
Sodium glycerophosphate	10.0
Maltose	20.0
Lactose	1.0
Sodium azide	0.4
Bromo-cresol purple	0.015
Agar	20.0
pH 7.2 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards

TTC SOLUTION (1%)

Code: SR0229

Vial contents (each vial is sufficient for 500ml of medium)
2,3,5-Triphenyltetrazolium chloride  (1%)	5ml

Directions
Suspend 38.2g in 500ml of distilled water. Bring to the boil with frequent agitation. Boil for 5 minutes.  Cool to 50°C and add aseptically 1 vial (5ml) of 1% 2,3,5-Triphenyltetrazolium chloride (SR0229K). Pour into sterile Petri dishes when using the membrane filtration method or hold at 45°C when using the pour-plate method.

Description
KF Streptococcus Agar is based on the formulation described by Kenner et al.¹ and is recommended² for the detection and enumeration of enterococci in faeces, milk, water and other materials by the pour-plate or membrane filtration method. The presence of enterococci in the material under test is indicative of faecal pollution by man or animals.

KF Streptococcus Agar Medium is selective for the following Group D and Group Q species.

Enterococcus group

Enterococcus faecalis	Group D
Enterococcus subsp. liquefaciens	Group D
Enterococcus faecalis subsp. zymogenes	Group D
Enterococcus faecium	Group D
Enterococcus bovis	Group D
Enterococcus equinus	Group D
Streptococcus avium	Group Q

Streptococcus avium (Group Q) has been included in the `enterococci’ group as it has very similar biochemical and antigenic characteristics to the Group D species and also occurs in warm-blooded animals.

The detection of enterococci may provide more specific information about the source of pollution because certain enterococci are host specific. For example, a predominance of Enterococcus bovis and Enterococcus equinus would indicate pollution due to animal excrement.

The detection of Enterococcus bovis and Enterococcus equinus species has been found to be associated with pollution involving meat processing plants, dairy wastes and feedlot and farmland run-off.

The detection of these enterococcal species is indicative of recent contamination as the organisms survive for only a short period outside their natural habitat. The coliform/enterococci ratios may also provide information on possible sources of pollution².

Caution must be observed when assessing the quality of marine recreational waters, particularly in tropical climates, because a high incidence of false-positive presumptive counts for enterococci may occur. Anaerobic incubation of tropical marine water samples is recommended³.

Colonies of enterococci on the membrane filter or agar plate are red or pink with a variation in diameter from 0.3 to 2mm. It is recommended that counting should be done with the aid of a low power (10-15 magnification) binocular wide field dissecting microscope or equivalent optical device.

Technique
Membrane Filtration Technique

1. Prepare the KF Streptococcus Agar Medium as directed.
2. Filter samples through a sterile membrane to give 20-100 colonies on the membrane surface. Use volumes of 100, 10, 1, 0.1 or 0.01ml, depending on the degree of pollution present.
3. Transfer the membrane filter directly to agar medium in the Petri dishes, avoiding the formation of air bubbles.
4. Invert the plates and incubate at 35°C for 48 hours.
5. Count all red or pink colonies with the aid of a low power (10-15 magnification) binocular wide field dissecting microscope.
6. Calculate the number of enterococci and report as faecal streptococci per 100ml.
7. Confirm the colonies as enterococci.

Plate Count Method

1. Prepare the KF Streptococcus Agar Medium as directed.
2. Prepare dilutions to give a count of 30-300 colonies. For most potable water samples plates suitable for counting will be obtained by inoculating 1ml and 0.1ml of undiluted sample and 1ml of sample diluted 1:100.
3. Measure the selected volume of sample into a Petri dish.
4. Pour 15ml of liquified medium into each plate.
5. Thoroughly mix the medium and sample to give a uniform dispersion of the organisms.
6. Solidify the agar as rapidly as possible after pouring
7. Incubate the plates in an inverted position at 35°C for 48 hours.
8. Count all red to pink surface and subsurface colonies.
9. Calculate the numbers of enterococci and report as faecal streptococci per 100ml.
10. Confirm the colonies as enterococci². Use an inoculating wire to stab through the agar to reach the colonies.

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 plates at 2-8°C.