MACCONKEY AGAR NO.3

Code: CM0115

A selective medium giving excellent differentiation between coliforms and non-lactose fermenters with inhibition of Gram-positive micrococci.

Typical Formula*	gm/litre
Peptone	20.0
Lactose	10.0
Bile salts No.3	1.5
Sodium chloride	5.0
Neutral red	0.03
Crystal violet	0.001
Agar	15.0
pH 7.1 ± 0.2 @ 25°C

* Adjusted as required to meet performance standards

Directions
Suspend 51.5g in 1 litre of distilled water. Bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes.

Description
A more selective modification of MacConkey medium which is suitable for the detection and enumeration of coliform organisms and also for the detection and isolation of Salmonella and Shigella species occurring in pathological and food specimens. Due to the inclusion of a specially prepared fraction of bile salts in addition to crystal violet, the medium gives improved differentiation between coliforms and non-lactose fermenting organisms whilst Gram-positive cocci are completely inhibited.

This formulation corresponds with that recommended by the American Public Health Association¹ for the direct plating of water samples for coliform bacilli, for the examination of food samples for food poisoning organisms² and for the isolation of Salmonella and Shigella species in cheese³.

Amongst other examples of the use of Oxoid MacConkey Agar No.3 are: the count of coli-aerogenes bacteria in poultry faecal specimens⁴; the count of coli-aerogenes bacteria in cattle and sheep faeces⁵; the count of coli-aerogenes and non- lactose fermenting organisms in poultry carcases⁶; bacterial counts on irradiated canned minced chicken⁷; the recognition of coli-aerogenes bacteria during investigations on the genus Aeromonas⁸.

Anderson et al ⁹ added 10mg/ml of kanamycin to MacConkey Agar to isolate epidemic strains of Citrobacter diversus which were causing neonatal meningitis.

The addition of 100mg of 4-methylumbelliferyl-ß-D-glucuronide to one litre of MacConkey Agar detects the enzyme ß-glucuronidase¹⁰. The cleaved 4-methylumbelliferyl moiety is fluorescent at 366nm. Thus colonies of Escherichia coli can be detected rapidly in mixed cultures by examining the plate under a UV lamp after overnight incubation at 35°C. However, it should be remembered that other organisms may also be ß-glucuronidase positive.

Technique
After inoculation the plates are usually incubated for 18 to 24 hours at 35°C and for a further 24 hours if non-lactose fermenting organisms are sought and have not appeared. Lower incubation temperatures may sometimes be used for more psychrophilic species. After 18 hours at 35°C, coliforms produce intense violet-red colonies whilst non-lactose fermenters are colourless.

Storage conditions and Shelf life
The dehydrated medium should be stored at 10-30°C and used before the expiry date on the label.
Store the prepared medium at 2-8°C.

Appearance
Dehydrated Medium: Straw coloured, free-flowing powder
Prepared medium: Dark red coloured gel

Quality control

Positive controls:	Expected results
Escherichia coli ATCC® 25922 *	Good growth; red colonies with bile precipitation
Shigella sonnei ATCC® 25931 *	Good growth; straw coloured colonies
Negative control:
Enterococcus faecalis ATCC® 29212 *	Inhibited