Which of the following types of agar differentiate lactose fermenters select all that apply

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.

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.

Toward 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, 10 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.

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 represents the views of the author and does not necessarily reflect the opinion of the American Society for Microbiology.

A culture medium is simply water and nutrients that support microbial growth. Primary culture media for clinical microbiology are divided into 3 primary categories — nutritive, differential and selective media. Media that support the growth of many different microorganisms without distinguishing genera or species are nutritive. In contrast, differential media allow several different types of bacteria to grow, but also contain compounds that allow microbial genera (or even species) to be visually differentiated. The organisms interact with the added compounds (e.g., blood, sugars) in ways that make them visually distinct amongst other bacterial types on a plate. This allows for the rapid identification of organisms of interest, which is especially important for heavily mixed cultures, such as stool.

Basic solid agar or liquid broth can also be enriched with various compounds specific for an organism of interest's needs (e.g. amino acids, vitamins, hormones). Selective media are used to select for the growth of a particular "selected" microorganism. For example, if a certain microbe is resistant to aparticular antibiotic (e.g., novobiocin), then that antibiotic can be added to the medium in order to prevent other organisms, which are not resistant, from growing. Likewise, other chemicals can be addedto media to create a selective environment. For example, NaCl media selects for halophiles (salt lovers) versus non-halophiles.

One of the most powerful applications of these types of media is combining both selective and differential characteristics into a single type of media. For example, mannitol salt agar is used to select for halophiles (e.g., Staphylococcus), while also visually differentiating species of staph based on mannitol fermentation (S. aureus) or the inability to ferment mannitol (S. epidermidis).

Microbiology Case Studies Using Differential & Selective Media

Case 1

A 28-year-old patient arrives at the emergency room with complaints of redness, pain and streaking along the site of a surgical incision on the leg. The patient has a fever, and the incision site is leaking purulent drainage. The patient is scheduled for surgical debridement of the wound, and during the procedure a tissue sample is collected and sent to the microbiology laboratory for culture. The specimen is plated to standard microbiology media, including blood agar (trypticase soy agar enriched with 5% sheep blood). After 24 hours of incubation, small colonies with a large zone of beta hemolysis are growing on the blood agar plate.

Explanation of Case 1

Blood agar is differential media because 3 different types of hemolysis, or lysing of red blood cells, can be seen on this plate. Blood agar allows for the growth of most types of bacterial organisms, but each organism’s ability to lyse red blood cells displays differently, which gives the microbiologist clues to its identification. Beta hemolytic organisms completely lyse red blood cells, leaving an area of total clearing underneath and around the colonies. Alpha hemolytic organisms partially lyse red blood cells, leaving the media a greenish color, while gamma hemolytic organisms do not lyse red blood cells at all and the media remains unchanged. When combined with patient history and colony morphology, hemolysis observed on blood agar can help microbiologists make a presumptive organism identification.

In this case, the patient’s sample grew Streptococcus pyogenes (Group A Streptococcus). Although additional testing is needed to confirm the identification, the characteristic small colonies with a large zone of beta hemolysis is highly suggestive of S. pyogenes, and knowing this helps guide the microbiologist toward the next best steps in identifying the organism. Additionally, the patient’s history is suggestive of necrotizing fasciitis, and the growth of S. pyogenes makes sense.

Case 2

The microbiology laboratory receives blood cultures on a 50-year-old patient with a variety of underlying, complex medical conditions. The blood cultures flag positive on the automated blood culture instrument at 18 hours of incubation, and gram-negative rods are seen on the Gram stain. The blood is plated to blood, MacConkey and chocolate agars and incubated for 24 hours. While the plates are incubating, the clinician calls the laboratory and says that the medical team is trying to choose appropriate antimicrobials for this patient, and they would like to know if the organism is Pseudomonas aeruginosa as soon as possible. The next day, pink colonies are growing on the MacConkey agar.

Source: Andrea Prinzi

Explanation for Case 2

MacConkey agar is an example of a medium that is both differential and selective. The presence of bile salts, as well as crystal violet, within the media prevent gram-positive organisms from growing. Furthermore, gram-negative rods can be differentiated between lactose fermenters and non-lactose fermenters based on the presence or absence of a pink color. When an organism metabolizes the lactose in the media, the surrounding agar becomes acidic and the neutral red pH indicator is activated, turning the colonies pink. If the organism does not ferment lactose, the colonies remain colorless.

Knowing if an organism is a lactose fermenter or not is incredibly helpful. For example, Pseudomonas aeruginosa does not ferment lactose, so pure growth of a lactose-fermenting, gram-negative rod in culture immediately rules out this organism. In the case above, the clinician can be told that the gram-negative rod growing in culture is not P. aeruginosa since it is a lactose fermenter on MacConkey agar. This information helps the clinician determine the best course of antimicrobial treatment because Pseudomonas has been ruled out, and the use of a broad spectrum antibiotic with antipseudomonal activity is not necessary.

Case 3

A 12-year-old patient presents to the emergency room with severe abdominal cramping and several days of watery diarrhea. A stool sample is collected and sent to the microbiology laboratory for culture.

Explanation of Case 3

The media used for stool culturing varies between laboratories, but stool testing is where the usefulness of differential media really shines. Typically, human feces contains a large variety of bacteria, and identifying pathogens amongst normal gastrointestinal flora can be challenging. Hektoen agar is a selective and differential medium that helps isolate and differentiate Salmonella and Shigella from normal enteric gram-negative organisms, while preventing the growth of most gram-positive organisms. This colorful medium contains 3 carbohydrates that differentiate fermentation characteristics among gram-negative organisms. The medium also includes sodium thiosulfate, which provides a source of sulfur. Non-pathogenic gastrointestinal flora will ferment the carbohydrates and produce bright salmon-colored colonies. Non-fermenting gram-negative rods, such as Shigella, will appear as blue-green colonies and organisms that reduce sulfur to hydrogen sulfide, such as Salmonella, will produce black colonies.

In the case above, the use of Hektoen agar allows the microbiologist to rapidly identify pathogens among a mass of normal flora. Being able to macroscopically identify pathogens like Salmonella saves time and tedious work for the microbiologist, and allows for the colony to be selected for subculture and isolated for further identification and susceptibility testing, if appropriate. Many other types of differential media can recover pathogenic bacteria from stool samples as well.

The Benefit of Differential & Selective Media, Even in the Time of Molecular Technology

Since the introduction in the 1970s of molecular diagnostics and techniques, including PCR, sequencing and, more recently, metagenomics, microbiologists and others in the world of microbial diagnostics have sometimes found these techniques to be superior to more traditional techniques. While there is a place for the powerful nature of these ever-evolving and important molecular diagnostic tools, the use of differential and selective media has specific advantages.

One obvious advantage for differential and selective media continues to be the cost of testing. Molecular diagnostic testing requires expensive reagents (e.g., enzymes), dedicated automation or semi-automated equipment, software and sometimes even specific personnel with the expertise to interpret results. Traditional media is less expensive and a foundational standard for medical laboratory and clinical microbiology majors.

Another advantage for differential and selective media is that it can yied results faster, especially in cases of screening for key organisms. In addition, molecular techniques (metagenomics) may not be as sensitive as the use of differential and selective media in some cases. For example, metagenomic primers lack the sensitivity to detect species present at concentrations <105 bacteria per gram of stool. Traditional use of differential and selective media also allows for the isolation of pure colonies of bacteria, which is strictly required for certain microbiological methods (e.g., susceptibility testing, biochemical testing and serotyping). Lastly, molecular techniques, while powerful in their ability to amplify DNA, cannot distinguish between living bacteria from transient or dead bacteria. For all these reasons, differential and selective media remain important tools in the clinical microbiology laboratory.