MacConkey Agar: Principle, Preparation & Colony Morphology

MacConkey Agar is one of the most widely used selective and differential media in microbiology. It plays a crucial role in identifying and differentiating Gram-negative enteric bacteria, particularly those belonging to the Enterobacteriaceae family and Pseudomonas species.

In this guide, we’ll explore the history, principle, preparation, colony characteristics, and uses of MacConkey agar, along with a table summarizing key bacterial examples and their appearances on MAC.

What is MacConkey Agar?

MacConkey Agar was developed in the early 20th century by Alfred Theodore MacConkey. It is a selective and differential medium specifically formulated for isolating and differentiating non-fastidious, Gram-negative rods, particularly those found in intestinal tracts.

Principle of MacConkey Agar

MacConkey Agar works on two main principles:

Selective Inhibition:
- Bile salts and crystal violet inhibit the growth of Gram-positive bacteria.
Differential Identification:
- Contains lactose as a fermentable carbohydrate.
- Includes neutral red as a pH indicator.
- Lactose fermenters produce acid, turning the medium pink to red.
- Non-fermenters remain colorless or pale.

MacConkey Agar Preparation

Here’s how to prepare 1 liter of MacConkey Agar:

Ingredients: Suspend 49.53 grams of MacConkey agar powder in 1000 mL of purified or distilled water.
Heating: Heat until completely dissolved and bring to a boil.
Sterilization: Autoclave at 121°C (15 psi) for 15 minutes.
Cooling: Cool to 45–50°C, then pour into sterile Petri dishes.

Storage: Store prepared media at 2–8°C, protected from light, moisture, and temperature fluctuations. Discard if you observe shrinking, discoloration, cracking, or signs of contamination.

Colony Characteristics on MacConkey Agar

MacConkey Agar helps differentiate bacteria based on their ability to ferment lactose:

Lactose Fermenters (Pink/Red Colonies)

Produce acidic by-products, lowering the pH and turning colonies pink or red.
Strong fermenters (like E. coli) may show a pink halo around colonies due to bile salt precipitation.

Lactose Non-Fermenters (Colorless Colonies)

Cannot ferment lactose; colonies remain colorless or pale.
Medium surrounding the colonies stays relatively transparent.

Composition of MacConkey Agar

Ingredient	Amount (g/L)	Function
Peptone (Pancreatic digest of gelatin)	17.0	Nitrogen source
Proteose peptone (meat)	3.0	Nitrogen source
Lactose monohydrate	10.0	Fermentable carbohydrate
Bile salts	1.5	Inhibits Gram-positive bacteria
Sodium chloride	5.0	Maintains osmotic balance
Neutral red	0.03	pH indicator
Crystal violet	0.001	Enhances Gram-negative selectivity
Agar	13.5	Solidifying agent

Bacteria on MacConkey Agar: Colony Appearance

Bacterium	Lactose Fermentation	Colony Appearance
Escherichia coli	Positive	Pink to red colonies
Klebsiella pneumoniae	Positive	Large, mucoid, pink colonies
Enterobacter aerogenes	Positive	Pink, slightly mucoid colonies
Salmonella typhi	Negative	Colorless or pale colonies
Proteus vulgaris	Negative	Colorless colonies

Uses of MacConkey Agar

Selective isolation of Gram-negative bacilli, especially coliforms and enteric pathogens.
Differentiation of lactose fermenters and non-fermenters among Gram-negative bacteria.
Widely used in clinical, food, and water microbiology to detect intestinal pathogens.

Limitations of MacConkey Agar

Not suitable for Gram-positive bacteria: They are inhibited and won’t grow.
Does not support fastidious Gram-negative bacteria (e.g., Haemophilus, Neisseria).
Colony color may vary depending on incubation time and light exposure.
Misidentification risk: Some non-lactose fermenters may look similar.

Conclusion

MacConkey Agar is a fundamental tool in microbiology laboratories for the isolation, identification, and differentiation of Gram-negative bacteria, especially from fecal, food, and environmental samples. Its ability to highlight lactose fermentation makes it a powerful diagnostic medium.