MacConkey Agar is used for the selective isolation and identification of Enterobacteriaceae from faeces, urine, wastewater, and foods. It can also be used as a selective and differential medium for the isolation of enteric Gram-negative bacteria. The specimen can be streaked directly on the medium or inoculated first into an enrichment broth like Tetrathionate Broth, Selenite Cystine Broth, GN Enrichment Broth, or MacConkey Broth. This media is recommended by the European Pharmacopoeia who recommends it is used for microbiological examinations of non-sterile products to test for specified microorganisms for the growth promoting and indicative properties of the media in the test for E. Coli. ISO 21567 lets this medium be used for the detection of Shigella spp. and is used to obtain isolated colonies. ISO 21150 also recommends the medium for the detection of E. Coli in cosmetic products.

After incubating the plates of Agar McConkey, a confirmatory step is necessary. Pancreatic digest of gelatine and peptones meat & casein provide nitrogen, vitamins, minerals, and amino acids that are essential for growth. Lactose is the fermentable carbohydrate providing carbon and energy, and sodium chloride supplies essential electrolytes for transport and osmotic balance. Bile salts and crystal violet are the selective agents that inhibit Gram-positive organisms. Neutral red is the pH indicator and when the lactose is fermented, the pH of the medium decreases which changes the colour of the neutral red to pink. Bacteriological agar is the solidifying agent, the isolated colonies of Escherichia coli are brick red in colour, and they are surrounded by a zone of precipitated bile. The bile precipitate is due to a local pH drop around the colony due to lactose fermentation.