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.
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.
- 1 Composition of MacConeky Agar
- 2 Preparation of MacConkey Agar
- 3 Principle behind differential capability of MacConkey agar
- 4 Intended use of MacConkey Agar:
- 5 Expected Colony characteristics in MacConkey Agar
- 6 Quality Control
- 7 Modifications of MacConkey Agar
Composition of MacConeky Agar
|Ingredient||MacConkey Agar (g/L)|
|Bile salts||1.5 g|
|Sodium chloride||5 g|
|Neutral red||0.03 g|
|Crystal violet||0.001 g|
|Distilled water||1 L|
- Enzymatic digest of Gelatin, Casein and Animal tissue: provides nitrogen, vitamins, minerals and amino acids essential for growth.
- Lactose: Lactose is the sole carbohydrate. Lactose-fermenting bacteria produce colonies that are varying shades of red, owing to the conversion of of the neutral red indicator dye (red below pH 6.8) from the production of mixed acids. Colonies of non-lactose fermenting bacteria appear colorless or transparent.
- Bile Salts: selective agents and inhibit Gram-positive organisms.
- Crystal Violet: Gram-positive bacteria are generally inhibited by crystal violet.
- Sodium Chloride: supplies essential electrolytes for transport and osmotic balance.
- 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
- Agar: Solidifying agent
Preparation of MacConkey Agar
- Suspend the measured amount of powder (See in the agar bottle and generally 50 gram) in 1 L of purified water and mix thoroughly.
- Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
- Autoclave at 121°C for 15 minutes.
You can purchase the ready-made MacConkey agar from the commercial suppliers.
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
- Typical strong lactose fermenters, such as species of Escherichia, Klebsiella, and Enterobacter produce red colonies surrounded by a zone of precipitated bile.
- Slow or weak lactose fermenters such as, Citrobacter, Providencia, Serratia, and Hafnia, may appear colorless after 24 hour or slightly pink in 24-48 hours.
- Non-lactose fermenters grow as colorless or transparent colonies e.g., species of Proteus, Edwardsiella, Salmonella, and Shigella.
Lactose Fermenter Organisms:
- 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.
- Klebsiella spp.: Mucoid lactose fermenter (MLF)
- Escherichia coli: Lactose fermenter; flat, dry, pink colonies with a surrounding darker pink area of precipitated bile salts.
- 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
- Proteus spp.: NLF; may swarm depending on the amount of agar in the medium; characteristic foul smell
- Shigella spp.: NLF; Shigella sonnei produces flat colonies with jagged edges.
- Yersinia spp.: NLF; may be colorless to peach.
- Salmonella spp.: NLF
- 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
- 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).
- Observe the MacConkey for specific colony morphology.
- As a sterility test, incubate an uninoculated plate for 48 hours at 35-37°C with ~5% CO2 (or in a candle-jar).
- E. coli should appear as pink to red colonies.
- After 48 hours, the sterility test plate should remain clear.
Modifications of MacConkey Agar
- 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.
- 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.
- 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
- Escherichia coli ATCC® 25922: Good growth, pink colonies are sorbitol positive
- Escherichia coli ATCC® 35150: Good growth, colorless colonies are sorbitol negative
- Quality control of Sorbitol MacConkey agar