MacConkey Agar (MAC): Composition, preparation, uses and colony characteristics

LF and NLF colonies in MacConkey Agar

This post was most recently updated on June 21st, 2019

MacConkey agar was developed in 20th century by Alfred Theodore MacConkey. It was the first formulated solid differential media. MacConkey agar is a selective and differential culture media commonly used for the isolation of enteric Gram-negative bacteria. It is based on the bile salt-neutral red-lactose agar of MacConkey.

Crystal violet and bile salts are incorporated in MacConkey agar to prevent the growth of Gram-positive bacteria and fastidious Gram-negative bacteria, such as Neisseria and Pasteurella. Gram-negative enteric bacteria can tolerate bile salts because of their bile-resistant outer membrane.

LF and NLF colonies in MacConkey Agar
LF and NLF colonies in MacConkey agar

MacConkey agar is selective for Gram-negative organisms and helps to differentiate lactose fermenting gram-negative rods from non-lactose fermenting gram-negative rods. It is primarily used for the detection and isolation of members of family enterobacteriaceae and Pseudomonas spp.

Composition of MacConeky Agar:

  1. Enzymatic digest of Gelatin, Casein and Animal tissue: provides nitrogen, vitamins, minerals and amino acids essential for growth.
  2. Lactose: fermentable carbohydrate providing carbon and energy.
  3. Bile Salts: selective agents and inhibit Gram-positive organisms.
  4. Crystal Violet: Gram-positive bacteria are generally inhibited by crystal violet.
  5. Sodium Chloride: supplies essential electrolytes for transport and osmotic balance.
  6. Neutral Red: pH indicator. which is red in color at pH below 6.8.
    When lactose is fermented, the pH of the medium decreases, changing the color of neutral red to pink
  7. Agar: Solidifying agent

Note: Remember the ingredient used in bold letter

Preparation of MacConkey Agar

  1. Suspend the measured amount of powder (See in the agar bottle and generally 50 gram) in 1 L 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.

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

LINK:  MacConkey Agar Plates (10/bx.)

Principle behind differential capability of MacConkey agar

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).
Strongly lactose fermenting bacteria produce sufficient acid which causes precipitation of the bile salts around the growth. It appears as a pink halo surrounding individual 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.

Intended use of MacConkey Agar:

MacConkey agar is used for the selective isolation and identification of Enterobacteriaceae from faeces, urine, wastewater and foods.

Expected Colony characteristics in MacConkey Agar

  1. Lactose-fermenting organisms grow as pink to brick red colonies with or without a zone of precipitated bile.
  2. Non-lactose fermenting organisms grow as colorless or clear colonies

Lactose Fermenter Organisms:

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
  1. Citrobacter spp.: Late lactose fermenter; therefore Non-Lactose Fermenter (NLF) after 24 hours; Lactose fermenter (LF) after 48 hours; colonies are light pink after 48 hours.
  2. Klebsiella spp.: Mucoid lactose fermenter (MLF)
  3. Escherichia coli: Lactose fermenter; flat, dry, pink colonies with a surrounding darker pink area of precipitated bile salts.
  4. Serratia spp.: Late lactose fermenter; S. marcescens may be red pigmented, especially if the plate is left at 25°C

Non-Lactose Fermenter (NLF) Organisms

  1. Proteus spp.: NLF; may swarm depending on the amount of agar in the medium; characteristic foul smell
  2. Shigella spp.: NLF; Shigella sonnei produces flat colonies with jagged edges.
  3. Yersinia spp.: NLF; may be colorless to peach.
  4. Salmonella spp.: NLF
  5. Other organisms showing colorless colonies on MacConkey agar are; Edwardsiella spp, Hafnia spp., Morganella spp., Providencia spp.

No Growth: Gram-positive bacteria

  • Staphylococcus aureus: No growth

QUALITY CONTROL

  1. Grow an E. coli quality control strain for 18-24 hours on a MacConkey agar at 35-37°C with ~5% CO2 (or in a candle-jar).
  2. Observe the MacConkey for specific colony morphology.
  3. As a sterility test, incubate an uninoculated plate for 48 hours at 35-37°C with ~5% CO2 (or in a candle-jar).

Passing result:

  • E. coli should appear as pink to red colonies.
  • After 48 hours, the sterility test plate should remain clear.

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 used also 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 and MacConkey Agar without 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
About tankeshwar 386 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.

18 Comments

  1. why do we used
    1-mac and blood agar plates for culture and sensitivity of sterile body fluids (CSF,pleural fluid,ascitic fluid,synovial fluid,pericardial fluid,blister fluid,sputum,BAL,ETT,TT)
    2-CLED agar for urine
    3- MRSA agar for nasal and groin sample

  2. can i have the references of this article ? i mean who is writing this article ? and are you quote this from the book or anything else ? Please answer me. Your article very important for my task.

  3. Excuse me sir, can i know is your real name? i found this website and want to use this post for my minithesis references

    • Dear Ayunda Novita Permatasari, My real name as well as short bio is available at the end of every post including these.

  4. Sir, is it possible for gram-positive bacteria to grow on macConkey agar plates as an exception? When we checked under the microscope, we found 2 different bacteria, which was coccus and rod. When we did gram staining, the bacteria was stained purple. We would really appreciate it if you could reply us as soon as possible.

    • Eugenie, MacConekey agar if contains bile salt and crystal violet, suppresses the growth of Gram Positive bacteria, but there are variant of MacConeky Agar available in the market. Please check the composition of the MacConkey you are using. If it does not contain those inhibitors, it will also support the growth of Gram-Positive bacteria.

    • Yes, you can. MacConkey Agar is selective for gram-negative bacteria and it differentiates between lactose fermenter and non-lactose fermenter. Salmonella and Shigella are NLF, that can cause GI infections but if the suspected organisms are other than these bacteria, culture media can be used accordingly.

      • Thanks very much for this post.
        Please which antibiotic can I add on Macconkey agar to make it further selective for Salmonella only rather than all the enterobacteriacae. (In other words want to inhibit the growth of other emterobacteriacae on Macconkey & permit only the growth of salmonella)

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