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Dehydrated Culture Media
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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.
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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
* This organism is available as a Culti-Loop®
Precautions
Prolonged incubation may lead to confusing results. Do not incubate beyond 48 hours.
Test the medium with a laboratory stock strain of Shigella species which is in the R-phase. R-phase
shigellae should grow satisfactorily on MacConkey Agar.
References
1. American Public Health Association (1998) Standard Methods for the Examination of Water and
Wastewater. 20th Edn. APHA Inc. Washington DC.
2. American Public Health Association (1976) Compendium of methods for the Microbiological
Examination of Foods. APHA Inc. Washington DC.
3. American Public Health Association (1978) Standard Methods for the Examination of Dairy
Products. 14th Edn. APHA Inc. Washington DC.
4. Barnes Ella M. and Goldberg H. S. (1962) J. Appl. Bact. 25(1). 94-106.
5. Medrek T. F. and Barnes Ella M. (1962) J. Appl Bact. 25(2). 159-168.
6. Barnes Ella M. and Shrimpton D. H. (1957) J. Appl. Bact. 20(2). 273-285.
7. Thornley Margaret J. (1957) J. Appl. Bact. 20(2). 273-285.
8. Eddy B. P. (1960) J. Appl. Bact. 23(2). 216-249.
9. Anderson R. L., Graham D. R. and Dixon R. E. (1981) J. Clin. Microbiol. 14. 161-164.
10. Trepeta A. W. and Edburg S. C. (1984) J. Clin. Microbiol. 19. 172-174.
11. Maddocks J. L. and Greenan M. J. (1975) J. Clin. Pathol. 28. 686-687.
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