Why Differential & Selective Media Remain Invaluable Tools

Sept. 25, 2020

Differential Media and Selective Media: What Are They and What's the Difference?

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.

Mannitol salt agar plate with clear and yellow colonies growing.
S. aureus is seen producing yellow colonies on mannitol salt agar. Notice non-mannitol fermenting clear colonies growing as well.

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.

Blood agar plate showing grow of alpha, beta and gamma hemolytic organisms.
From left to right: alpha, beta and gamma hemolytic organisms growing on blood agar.

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.

Two MacConkey agar plates, the left plate is growing a lactose-fermenting organism as pink colonies, while the right is growing a non-lactose fermenter as clear colonies.
Left: MacConkey agar growing a lactose-fermenting, gram-negative rod, which produces a pink pigment. Right: MacConkey agar growing a non-lactose fermenter, which displays colorless colonies.
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.

Hektoen agar with black colonies of Salmonella growing.
Salmonella growing as black colonies on Hektoen agar.

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.

Author: Andrea Prinzi, Ph.D., MPH, SM(ASCP)

Andrea Prinzi, Ph.D., MPH, SM(ASCP)
Andrea Prinzi, Ph.D., MPH, SM(ASCP) is a field medical director of U.S. medical affairs and works to bridge the gap between clinical diagnostics and clinical practice.

Author: Rodney Rohde, Ph.D., SM(ASCP), SVCM, MBCM, FACSc

Rodney Rohde, Ph.D., SM(ASCP), SVCM, MBCM, FACSc
Dr. Rodney Rohde is the Associate Director of the Translational Health Research Initiative at Texas State University.