The Origin of MacConkey Agar

Oct. 14, 2019

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 “ordinary earth organisms.” Not surprisingly, his samples often grew large numbers of colonies on standard nutrient media. Despite dilution, it proved difficult to identify enterics that may have been present.
 

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 amphipathic molecules found in the gut that aid in digestion by emulsifying fats and allowing them to be transported in an aqueous environment.
 
Cellular membranes also look very much like fats, so bile acids are toxic to many organisms through disruption of this barrier. Enteric organisms, however, must withstand a constant assault from bile acids in the gut and have thus evolved mechanisms to resist their action. Therefore, enterics (and a select group of other Gram-negative bacteria, notably Pseudomonas) are selected for on media containing bile.

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 Escherichia coli (then Bacillus coli communis) or Salmonella enterica serovar Typhi (then B. typhi abdominalis). Although definitive identification of these organisms required additional testing, MacConkey used the previous observation by Theodor Escherich (for whom the genus Escherichia is named) that E. coli ferments the sugar lactose whereas Salmonella does not ferment lactose to quickly rule in or rule out these organisms by sight.
 
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 litmus). However, if broth-based assays contained more than one organism, as would often be the case for MacConkey’s water samples, any fermenting bacteria would drop the pH and obscure presence of non-fermenting organisms.
 
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 first description of MacConkey agar was published in The Lancet in 1900, use of the medium caught on rapidly amongst those interested in water microbiology. However, other scientists recognized that MacConkey’s original recipe had some limitations. One of these was the difficulty of evaluating lactose fermentation in organisms that that did not produce enough acid during the fermentation process to precipitate bile. To address this issue, Albert Grunbaum and Edward Hume added neutral red, a pH indicator that transitions from yellow at basic pH to red at acidic or neutral pH. This addition allowed for greater sensitivity of detecting lactose fermentation.
 
Another limitation was that bile was the sole selective agent, allowing growth of bile-resistant Gram-positive organisms. Grunbaum and Hume’s modification additionally contained crystal violet, a dye that Wilhelm von Drigalski and Heinrich Conradi had previously shown to be inhibitory towards Gram-positives. This addition was important to increasing selectivity to exclude Enterococcus spp.

Modern Day MacConkey Agar

By 1930, ten modifications of “MacConkey’s Basal Bile Salt Peptone” agar were published in a compendium of microbiological media. These included variations in bile content, substitution of lactose for other sugars, changes in pH indicator, or addition of inorganic salts. Among all of these, it was Grunbaum and Hume’s formula that stood the test of time and is (with minor modifications) the basis of modern 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 Food and Drug Administration’s Bacteriological Analytical Manual (BAM) as an important tool for water quality testing. Despite foundational changes in microbiology practice, including automation, molecular genetics, and mass spectrometry, it seems likely that MacConkey’s medium will continue to be used in the foreseeable future.

The above represent the views of the author and does not necessarily reflect the opinion of the American Society for Microbiology.

Author: Kenneth (K.P.) Smith

Kenneth (K.P.) Smith
Dr. Kenneth (K.P.) Smith is a postdoctoral fellow at Beth Israel Deaconess Medical Center. His research interest is in development of drugs and diagnostics for multidrug-resistant gram-negative bacteria.