In the late 1890’s, Alfred MacConkey was working at the University of Liverpool under the auspices of the Royal Commission on Sewage Disposal. This group was charged with protecting the public from waterborne disease through developing best practices for treatment of sewage. To evaluate the efficacy of various sewage treatment regimens, the commission’s work necessarily involved determining whether treated water remained contaminated by feces.

Part of MacConkey’s role on the commission was to survey drinking water sources for the presence of Gram-negative enteric organisms. These bacteria are normal inhabitants of the gastrointestinal tract of humans and are also found in other mammals, reptiles, and birds. Although they do not always cause disease themselves, their presence is an indicator of fecal contamination and therefore, the potential presence of other fecally transmitted pathogens.

To identify enteric organisms, water samples were plated on solid media and the colonies that formed were enumerated and identified. However, MacConkey’s efforts were frustrated by the fact that every milliliter of treated water may still contain hundreds or thousands of bacteria. Many of these are environmental organisms were not predictive of contamination, what MacConkey called

MacConkey Agar is Selective for Non-fastidious Gram-negative organisms

Therefore, MacConkey needed a way to limit this background of environmental flora and allow only his organisms of interest to grow. A medium that can perform this function is now known as a selective medium. His strategy for selection of enteric organisms was to add bile acids to standard media. Bile acids are

Cellular membranes also look very much like fats, so bile acids are

MacConkey Agar Differentiates Lactose fermenters and Non-fermenters

In addition to enriching for Gram-negative bacteria, MacConkey also wanted to be able to differentiate between types of enteric organisms. Of particular interest was determining whether a colony represented

This medium was made using modern bacteriological media components according to MacConkey’s original formulation, published in the Lancet in 1900. Pure deoxycholic acid replaced the mixture of glycholic acid and taurocholic acid originally used by MacConkey. Panel A shows Escherichia coli, a lactose fermenter. The white color surrounding the colony represents precipitation of bile. Panel B shows Klebsiella pneumoniae. Although this organism also ferments lactose, it does not produce sufficient acid to precipiate bile and looks like a non-fermenter on this medium. Panel C shows Pseudomonas aeruginosa, a lactose non-fermenter. Source: courtesy K.P. Smith

When bacteria ferment a sugar, the pH of the medium becomes acidic. Of course, acidity cannot be directly observed, so sugar fermentation was traditionally assayed in broth media containing a chemical pH indicator (often

What MacConkey needed was a way to evaluate lactose fermentation on individual colonies on solid media. To do this, he incorporated lactose directly into the agar. Changes in pH attributable to fermentation were observed by taking advantage of the knowledge that bile acids precipitate in an acidic environment. In this way, lactose-fermenting colonies were surrounded by a haze of precipitated bile.

Towards the Modern Formulation of MacConkey Agar

After the

Another limitation was that bile was the sole selective agent, allowing growth of bile-resistant Gram-positive organisms

Modern Day MacConkey Agar

Modern, commercially available MacConkey agar. Panel A shows Escherichia coli, a lactose fermenter. Note the opaque pink bile precipitation around the colonies. Panel B shows Klebsiella pneumoniae, also a lactose fermenter. Colonies are pink, indicating acid production but bile precipitation is absent. Panel C shows Pseudomonas aeruginosa, a lactose non-fermenter. Source: courtesy K.P. Smith

Almost 120 years later, MacConkey agar remains ubiquitous in clinical laboratories, where it is used routinely to select for non-fastidious Gram-negative organisms in wound, urine, stool, and blood cultures. Additionally, it is recognized in the