MacConkey Agar: Composition, Uses, Colony Characteristics

Last updated on June 21st, 2021

MacConkey agar is a selective and differential culture media commonly used for the isolation of enteric Gram-negative bacteria.  It is selective as it allows the growth of gram-negative bacteria and differential as it differentiates the gram-negative bacteria based on their lactose metabolism. MacConkey agar is used for the selective isolation and identification of members of the family Enterobacteriaceae from feces, urine, wastewater, and foods.

The media was first developed by Alfred Theodore MacConkey in 20th century.

Key components of the MacConkey medium include crystal violet dye, bile salts, lactose, and neutral red (pH indicator), all of which have a unique property.

Colony characteristics in MacConkey Agar

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 utilized 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.

Composition of MacConkey Agar

Ingredient MacConkey Agar (g/L)
Peptone17 g
Polypeptone3 g
Lactose10 g
Bile salts1.5 g
Sodium chloride5 g
Agar13.5 g
Neutral red0.03 g
Crystal violet0.001 g
Distilled water1 L
Final pH7.1

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 proliferate the selective growth of gram-negative bacteria. Lactose acts as a source of carbohydrate. 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.

Mixed growth of mucoid Lactose fermenting colonies and NLF colonies in MacConkey Agar
Mixed growth of mucoid lactose fermenting colonies and NLF colonies in MacConkey agar

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

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

Dry pink colonies of E. coli in MacConkey Agar
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. Pink halo is not seen around the colonies of weaker lactose fermenting bacteria.

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.

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

Preparation of MacConkey Agar

  1. Weigh and suspend 50 grams of MacConkey agar powder (see the instruction of the manufacturer) in 1 Litre of purified water and mix thoroughly.
  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 and pour about 20- 25ml into sterile Petri plates and allow to solidify.
  5. After solidification of the plates, label the media plates with name and date of preparation (on the backside of the media as lids could be interchanged)
  6. Store inverted (with lids down) at 2-8°C until use.

You can also purchase the ready-made MacConkey agar from the commercial suppliers.

Result and Interpretation

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

Pale (NLF) and pink (LF) colonies on MacConkey Agar
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 indicate that the test organism is a non-lactose fermenter. Examples include species of Salmonella, Shigella, Proteus, Providencia, Pseudomonas, Morganella, etc.

Colony Morphology of some common bacteria on MacConkey Agar

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

OrganismTypeDifferentiating Colonial characteristics
Escherichia coliLactose fermenterFlat, dry, pink, non-mucoid colonies with a surrounding darker pink area of precipitated bile salts.
Klebsiella sppLactose fermenterColonies typically appear large, mucoid, and pink, with pink- red pigment usually diffusing into the surrounding agar
Citrobacter sppLate lactose fermenterAppear as non-lactose fermenter (NLF) up to 24 hours; however, after 48 hours colonies are light pink.
Enterobacter sppLactose fermenterPink, mucoid colonies but smaller than Klebsiella spp.
Serratia sppLate lactose fermenterS. marcescens may be red-pigmented, especially if the plate is left at 25°C
Proteus sppNon-Lactose FermenterPale colonies with swarming, characteristic foul smell.
Shigella sppNon-Lactose Fermenter except S.sonnei which is a late lactose fermenterPale colonies,1-2 mm, flat colonies with jagged edges
Providencia sppNon-Lactose FermenterColonies are colorless, flat, 2-3mm in diameter and do not swarm.
Salmonella sppNon-Lactose FermenterColorless colonies,convex,2-3 mm with serrated margin
Pseudomonas sppNon-Lactose FermenterColorless, flat, smooth colonies, 2-3mm in diameter with greenish to brownish pigmentation.
Yersinia sppNon-Lactose FermenterColonies may be colorless to peach
Gram positive bacteria
No growth
Pink-mucoid colonies of Klebseilla pneumoniae in MacConkey Agar
Pink-mucoid colonies of Klebseilla pneumoniae in MacConkey Agar

In MacConkey agar without Crystal violet and bile salts

Staphylococcus sppLactose fermenterSmall pink colonies, 1-2mm in diameter, opaque
Enterococcus sppLactose fermenterDark pink to red, very minute, translucent colonies

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 of MacConkey agar

  1. Sterility testing: Incubate uninoculated plates of Mac conkey agar 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 mac conkey agar plates, incubate for 18-24 hours at 35-37°C and observe for growth and colony characteristics.
OrganismUsed asColony morphology
Escherichia coli ATCC 25922Positive control for lactose fermentationLactose fermenting pink colonies
Pseudomonas aeruginosa ATCC 27853Positive control for non- lactose fermentationNon lactose fermenting colonies with or without pigmentation.
Streptococcus pneumoniae ATCC 49619Negative controlNo growth

Modifications of MacConkey Agar

  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 (VTEC) 0157 is non-sorbitol fermenting, producing 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

References and further readings

  • Allen, M. E. (2005, September 30). MacConkey Agar Plates Protocols. https://www.asmscience.org/content/education/protocol/protocol.2855
  • Jung, B., & Hoilat, G. J. (2021). MacConkey Medium. In StatPearls. StatPearls Publishing. http://www.ncbi.nlm.nih.gov/books/NBK557394/
About Acharya Tankeshwar 476 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.