MacConkey Agar: Composition, Principle, Preparation, Uses, and Colony Characteristics

MacConkey agar (MAC) is the most widely used selective and differential medium in clinical bacteriology. It was developed in 1900 by British bacteriologist Alfred Theodore MacConkey, who originally designed it to differentiate typhoid bacilli (Salmonella Typhi) from coliform organisms contaminating water supplies; a critical public health need at the turn of the 20th century. More than 120 years later, it remains a first-line plating medium in virtually every diagnostic microbiology laboratory in the world.

MacConkey agar (MAC) is a selective and differential bacterial culture media. MacConkey medium is selective for gram-negative bacteria and differentiates the gram-negative bacteria based on lactose metabolism.

This medium was first developed by Alfred Theodore MacConkey in 1900.

Figure: Colony characteristics in MacConkey Agar

Composition of MacConkey Agar

Key components of the MacConkey medium include crystal violet dye, bile salts, lactose, and neutral red (pH indicator).

Crystal violet dye and bile salts prevent the growth of Gram-positive bacteria and fastidious Gram-negative bacteria (such as Neisseria and Pasteurella) making it favorable for the growth of gram-negative bacteria. Since Gram-negative enteric bacteria possess a bile-resistant outer membrane, they remain unaffected by bile salts.

Lactose present in the medium is fermented by bacteria to form lactic acid that decreases the pH of the agar, and turns the indicator (neutral red) pink, thus differentiating lactose fermenters from non-lactose fermenters.

Other ingredients such as enzymatic digest of gelatin, casein, and animal tissue provide nitrogen, vitamins, minerals, and amino acids essential for growth. Sodium chloride provides osmotic balance and supplies essential electrolytes for transport. Agar is incorporated as the solidifying agent.

Principle of MacConkey Agar

MacConkey agar contains four key ingredients (lactose, bile salts, crystal violet, and neutral red) that make it a selective and differential media.

Bile salts and crystal violet act as selective agents that inhibit the growth of Gram-positive organisms, and aid in the selective growth of non-fastidious gram-negative bacteria.

Lactose acts as a source of carbohydrates. Lactose-fermenting bacteria produce pink-red colonies, after fermenting the lactose to acids and dropping the pH of the indicator (neutral red) present in the medium. Since, non-fermenters can’t utilize lactose, colonies appear colorless or transparent.

Encapsulated bacteria such as Klebsiella and Enterobacter produce capsules using lactose. This gives sticky, wet-appearing colonies on MacConkey medium.

Figure: Mixed growth of mucoid lactose fermenting colonies and NLF colonies in MacConkey agar

Lactose Fermenters

Gram-negative enteric bacteria that grow on MacConkey medium are differentiated by their ability to ferment lactose. If the lactose is fermented the production of the acid drops the pH of the media. The drop in pH is indicated by the change of the neutral red indicator to pink (neutral red appears pink at pH below 6.8).

Figure: Dry pink colonies of E. coli in MacConkey Agar

Strongly lactose fermenting bacteria produce sufficient acid which causes precipitation of the bile salts around the growth. It appears as a pink halo surrounding colonies or areas of confluent growth. A pink halo is not seen around the colonies of weaker lactose fermenting bacteria.

Lactose non-fermenters

Gram-negative bacteria that grow on MacConkey agar but do not ferment lactose appear colorless on the medium and the agar surrounding the bacteria remains relatively transparent.

Figure: Pale, non-lactose fermenting colonies of Salmonella in MacConkey Agar

Preparation of MacConkey Agar

1. Weigh and suspend 50 grams of MacConkey agar powder in 1 Liter of purified water and mix thoroughly. Read and follow the instruction of the manufacturer if you have purchased dehydrated agar media from a commercial supplier.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. Cool to 45-50°C, mix well, pour about 20- 25mL into sterile Petri plates and allow to solidify.
5. After solidification of the plates, label the media plates with the name and date of preparation. Labeling should always be done on the backside of the media plate, as lids could be interchanged.
6. Store inverted (with lids down) at 2-8°C until use.

Ready-made MacConkey agar plates are also available from commercial suppliers.

Inoculation and Incubation

1. Allow the plate to reach room temperature and dry the agar surface before use.
2. Inoculate directly from the specimen. Suitable specimens include urine, stool, wound and pus swabs, sputum and respiratory samples, and positive blood culture subcultures.
3. If culturing from a swab, roll the swab over a small area at the edge of the plate first, then streak out from that area for isolation with a sterile loop.
4. Incubate aerobically at 35 to 37°C. MacConkey does not require carbon dioxide, and CO₂ can slightly acidify the medium, so incubate in air.
5. Read the plate at 18 to 24 hours for the primary result, then read again at 48 hours. This second read is not optional: late lactose fermenters such as Citrobacter, Serratia, and Shigella sonnei can appear colorless at 24 hours and turn pink only by 48 hours. Reporting at 24 hours alone is the most common way a fermenter is misclassified as a non-fermenter.
6. For suspected Yersinia enterocolitica, incubate at 25 to 28°C for 48 to 72 hours, because it is recovered better at room temperature than at 37°C.

Result and Interpretation

Pink-red colonies: Pink-red colonies on MAC medium indicate the presence of lactose fermenting bacteria. Examples include Escherichia coli, Klebsiella spp, Citrobacter, Enterobacter, etc.

Figure: Pale (NLF) and pink (LF) colonies on MacConkey Agar

Colorless colonies/pale colonies (colonies similar to the color of the media): Colorless or pale colonies on MAC medium indicate that the test organism is a non-lactose fermenter. Examples include species of Salmonella, Shigella, Proteus, Providencia, Pseudomonas, Morganella, etc.

Uses of MacConkey Agar Medium

1. Urine culture

MacConkey agar is routinely used for urine culture. Urine specimens contain a mix of urinary pathogens and contaminating organisms; MacConkey selects for gram-negative uropathogens while suppressing gram-positive contamination. It is typically used alongside blood agar and CLED agar in a standard urine culture setup.

2. Stool culture for enteric pathogens

MacConkey agar is a primary plating medium for fecal specimens when enteric pathogens such as Salmonella, Shigella, Campylobacter, and E. coli O157:H7 are suspected. The ability to rapidly differentiate lactose fermenters (normal gut flora like E. coli) from non-lactose fermenters (potential pathogens like Salmonella and Shigella) at 24 hours guides the laboratory directly toward the likely pathogen.

3. Wound, pus, and respiratory specimens

MacConkey agar is included in the routine workup of wound swabs, pus, sputum, tracheal aspirates, and bronchial washings, particularly when gram-negative organisms such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, or members of Enterobacteriaceae are suspected clinically.

4. Blood culture subcultures

When blood culture bottles flag positive, the broth is subcultured to a battery of media including MacConkey agar. The colony characteristics at 24 hours (color, size, mucoid appearance, pigmentation) provide the first presumptive identification while awaiting further biochemical or MALDI-TOF results.

5. Differentiation of Enterobacteriaceae

MacConkey agar is foundational for separating members of the Enterobacteriaceae family based on lactose fermentation speed and colony appearance.

Strong fermenters (E. coli, Klebsiella), late fermenters (Citrobacter, Serratia), and non-fermenters (Salmonella, Shigella, Proteus) are distinguished at 18–48 hours without any additional testing.

6. Water and environmental microbiology

MacConkey agar is used in water quality testing to detect and enumerate coliforms: gram-negative, lactose-fermenting rods that indicate fecal contamination of water. The presence of pink lactose-fermenting colonies on MacConkey agar is the basis for the presumptive coliform test in standard water analysis.

7. Food microbiology

MacConkey agar is approved by the Bacteriological Analytical Manual (FDA-BAM) for the detection of gram-negative pathogens in food products, particularly for Salmonella and coliforms in meats, dairy, and processed foods.

8. Detection of E. coli O157:H7 (Sorbitol MacConkey)

The sorbitol variant of MacConkey agar (SMAC) replaces lactose with sorbitol and is specifically used to detect E. coli O157:H7, the cause of hemorrhagic colitis and hemolytic uremic syndrome (HUS). Unlike most E. coli strains, O157:H7 does not ferment sorbitol and therefore appears as colorless colonies on SMAC, while other E. coli produce pink colonies.

Colony Morphology on MacConkey Agar

Besides differentiating on the basis of color, colonies on MacConkey medium can further be presumptively identified based on their colonial appearances (shape, size, margin, time of growth, etc). Some of them are enlisted below:

Lactose fermenters (pink/red colonies)

Non-lactose fermenters (colorless/pale colonies)

Figure: Pink-mucoid colonies of Klebsiella pneumoniae in MacConkey Agar

In MacConkey agar without crystal violet and bile salts

Figure: Light pink, small colonies of S. aureus (above) and dark pink-red, minute colonies of Enterococcus (below) on MacConkey agar without bile salts and crystal violet.

Note: Gram-positive organisms are inhibited in MacConkey agar with bile salts and crystal violet, however, in a different formulation where bile salt and crystal violet are not incorporated, Gram-positive organisms also appear as lactose fermenters but are smaller in size than gram-negative ones.

Quality Control

1. Sterility testing: Incubate uninoculated plates of MacConkey for 48 hours at 35-37°C and observe for any growth. After 48 hours, the sterility test plate should remain clear. Discard the whole lot if any colonies are seen.
2. Performance testing: Inoculate known standard strains on MacConkey agar plates, incubate for 18-24 hours at 35-37°C, and observe for growth and colony characteristics.

Modifications

1. MacConkey Agar without Crystal Violet It is a differential medium but is less selective than MacConkey agar. The lack of crystal violet permits the growth of Staphylococcus and Enterococcus. Staphylococci produce pale pink to red colonies and enterococci produce compact tiny red colonies either on or beneath the surface of the medium. The medium is also used to separate Mycobacterium fortuitum and M. chelonae from other rapidly growing mycobacteria.
2. MacConkey Agar, CS (“Controlled Swarming”):  MacConkey agar without crystal violet or salt is used to prevent the swarming of Proteus spp.
3. Sorbitol MacConkey Agar: Sorbitol MacConkey agar is a variant of MacConkey agar, it contains sorbitol instead of lactose as fermentable sugar. The contents of Sorbitol MacConkey agar are sorbitol, peptone, bile salts, sodium chloride, neutral red, crystal violet, and agar. E. coli O157:H7 (a verotoxigenic strain) is non-sorbitol-fermenting and produces colorless colonies. Most other E. coli strains and other enterobacteria ferment sorbitol. Sorbitol-fermenting organisms produce pink colonies

Quality control of Sorbitol MacConkey agar

1. Escherichia coli ATCC® 25922: Good growth, pink colonies are sorbitol positive
2. Escherichia coli ATCC® 35150: Good growth, colorless colonies are sorbitol negative.

How to Remember

• Pink means acid, pale means none. On MacConkey, color is a pH report. Lactose fermenters make acid, the neutral red turns them pink; non-fermenters make no acid and stay the color of the medium. If you remember only one thing, remember that the plate is reading pH, not counting sugar.
• The halo means "strong." A pink halo of precipitated bile salts around a colony means so much acid was produced that the bile salts fell out of solution. Only vigorous fermenters like E. coli do this, so the halo is a quick "strong fermenter" flag in a mixed plate.
• Two selective agents, one job: keep Gram-positives out. Bile salts and crystal violet are the doormen. Take them away (MacConkey without crystal violet) and Gram-positives like Staphylococcus and Enterococcus walk in. This is why the "without crystal violet" formulation exists.
• Late fermenters are the trap. Citrobacter, Serratia, and Shigella sonnei can look colorless at 24 hours and pink by 48. Reading a plate too early is the classic way a fermenter gets misfiled as a non-fermenter.
• Mucoid and stringy = capsule. Klebsiella looks wet and mucoid and strings when you touch it with a loop, because it pours lactose into capsule production. Mucoid pink is a Klebsiella signature.

Key exam facts

Where Students Get Confused

• Colorless does not always mean non-fermenter. Late lactose fermenters (Citrobacter, Serratia, Shigella sonnei, Hafnia) look colorless at 24 hours and turn pink by 48 hours. Always confirm a "non-fermenter" with a 48-hour read before reporting it.
• Salmonella and Shigella look identical on MacConkey. Both are colorless non-fermenters. Color cannot separate them. Salmonella is convex and 2 to 3 mm; Shigella is flatter, smaller, with jagged edges, but this is only a hint. Definitive separation needs biochemical testing or serology.
• Mucoid colorless colonies are not always Klebsiella. Mucoid Pseudomonas aeruginosa from cystic fibrosis isolates can look like mucoid Klebsiella, but Klebsiella is a pink lactose fermenter and mucoid Pseudomonas is a colorless non-fermenter. Check the color and the pyocyanin pigment before assuming Klebsiella.
• Acinetobacter can look faintly pink. Acinetobacter is a non-lactose fermenter and should be colorless, but light refraction can give it a faint lavender-pink tint that is mistaken for weak fermentation. It is oxidase negative and non-motile; do not read the refraction as fermentation.

References and further readings

1. Tille, P. M. (2022). Bailey & Scott's Diagnostic Microbiology (15th ed.). Elsevier.
2. Procop, G. W., Church, D. L., Hall, G. S., Janda, W. M., Koneman, E. W., Schreckenberger, P. C., & Woods, G. L. (2017). Koneman's Color Atlas and Textbook of Diagnostic Microbiology (7th ed.). Wolters Kluwer.
3. Jung, B., & Hoilat, G. J. (2023). MacConkey Medium. In StatPearls. StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK557394/
4. Public Health England. (2015). Identification of Enterobacteriaceae (UK Standards for Microbiology Investigations, ID 16). https://www.gov.uk/government/publications/smi-id-16-identification-of-enterobacterales
5. Atlas, R. M., & Snyder, J. W. (2014). Handbook of Media for Clinical and Public Health Microbiology. CRC Press.