MacConkey Agar: MAC

MacConkey agar is also known as MAC. It is a bacterial culture medium that is named after bacteriologist Alfred T. MacConkey (1861-1931). MacConkey agar is a selective and differentiating agar of non-fastidious gram-negative rods, particularly members of the family Enterobacteriaceae and the genus Pseudomonas. It further differentiates gram-negative organisms based on their lactose metabolism. The selective and differentiating properties of MacConkey agar facilitates the utilization for both research and clinical applications. The fermentation of lactose production is organic acids, particularly lactic acid, which decreases the pH of the agar. Under acidic conditions, MAC consist of a pH indicator that turns pink. Thus, lactose-fermenting-gram-negatives (lactose-fermenters) will form pink colonies, while non-lactose fermenters will form off-white opaque colonies. Even within lactose-fermenters, species will show a fluctuating rate of growth. The rate of growth is also a way to differentiate organisms in the MAC medium.

Also read- Morphology: Size, shape and arrangement of Bacteria (mybiologydictionary.com)

Lastly, some species that form a capsule show up differently. Generally, MacConkey agar only becomes gram-negative bacteria, and those bacteria will appear separately based on their lactose fermenting ability as well as the rate of fermentation and the existence of a capsule or not. This makes MAC a powerful tool for differentiating and isolating bacterial species from the sample source. MAC is one of the many bacterial cultures clinical microbiologists utilize for diagnostic testing. It is still widely used in the clinical laboratory to identify causal agents from a patient (i.e., stool sample).

Lac positive:

By using the lactose available in the medium, Lac+ bacteria such as Escherichia, Enterobacter and Klebsiella will produce acid, which decreases the pH of the agar below 6.8 and as result give the appearance of pink colonies. The bile salts precipitate in the immediate section of the colony, causing the medium surrounding the colony to become hazy.

Lac negative:

Organisms not able to ferment lactose will form normal-coloured that is undyed colonies. The medium will remain yellow. For example, non-lactose fermenting bacteria are Salmonella, Proteus species, Yersinia, Pseudomonas aeruginosa, and Shigella. 

Slow:

There are some organisms that ferment lactose slowly or weakly and are sometimes put in their class. These include Serratia and Citrobacter.

Mucoid colonies:

Some other organisms, especially Klebsiella and Enterobacter, produce mucoid colonies which develop as very moist and sticky, and slimy. This event happens because the organism is producing a capsule, which is mainly made from the lactose sugar in the agar.

History Of MacConkey Agar:

MacConkey agar was the first solid differential media to be formed. It was advanced at the turn of the 20th century by Alfred Theodore MacConkey. After that Assistant Bacteriologist to the Royal Commission on Sewage Disposal, in the Thompson-Yates Laboratories of Liverpool University, England. The main target was to formulate a medium that would select for the growth of gram-negative microorganisms and prohibit the growth of gram-positive microorganisms.
In 1900, Dr. MacConkey first developed a bile salt medium that contain glycocholate, lactose, and litmus. This medium should be incubated at 22°C. In 1901, this formula was soon modified by the replacement of glycocholate with taurocholate, and the incubation temperature was raised to 42°C. Again in 1905, he later changed the method by substituting neutral red for litmus. In 1902, another suggestion was given by Grunbaum and Hume that neutral red can be used as an indicator in a bile salt lactose medium. The final media production was designed to support or back the growth of Shigella and is the one that is most commonly used today.

Principle Of MacConkey Agar:

MacConkey agar is mainly used for the isolation of gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non-fermenting gram-negative bacteria. It has also become common to use the media to differentiate or separate bacteria by their abilities to ferment other than lactose-like sugar. These altered media are used to differentiate gram-negative bacteria or to distinguish between phenotypes with mutations that consult varying abilities to ferment particular sugars.

Pancreatic digest of gelatin and peptones which consists of meat and casein. These provide the essential nutrients, vitamins, and nitrogenous factors required for the growth of microorganisms. The fermentable source of carbohydrates is the Lactose monohydrate. The selective action of this medium is associated with crystal violet and bile salts, which act as inhibitors to most species of gram-positive bacteria. Sodium chloride manages or maintains the osmotic balance in the medium. Neutral red is a pH indicator that turns red when the pH is below 6.8. It is white or colourless at any pH greater than 6.8. Agar is act as the solidifying agent.

Composition Of MacConkey Agar:

The composition of MacConkey Agar is as follows with the amount and ingredients:

• 17 gm of peptone is a pancreatic digest of gelatin
• 3 gm of peptone which is a meat and casein
• 10 gm of lactose monohydrate
• 1.5 gm of Bile salts
• 5 gm of Sodium chloride
• 0.03 gm of Neutral red
• 0.001 gm of Crystal Violet
• 13.5 gm of Agar
• Add distilled water to make 1 litre

Preparation Of MacConkey Agar :

1. 49.53 grams of dehydrated medium suspended in 1000 ml purified or distilled water.
2. After that heat to boiling to dissolve the medium completely.
3. To sterilize the medium we autoclave at 15 lbs pressure (121°C) for 15 minutes.
4. Then, cool to 45-50°C.
5. Before pouring the media into sterile Petri plates mix well.

Result Analysis on MacConkey Agar:

Lactose fermenting strains become as red or pink. It may be enclosed by a zone of acid-precipitated bile. Due to the production of acid from lactose, its colour is red. Absorption of neutral red and a consequent colour change of the dye when the level of pH of the medium falls below 6.8.

Lactose non-fermenting strains, such as Shigella and Salmonella are colourless or dull and transparent or translucent. It typically does not change the appearance of the medium. Yersinia enterocolitica may show up as small, non-lactose fermenting colonies after incubation at room temperature.

Uses of MacConkey Agar:

1. MacConkey agar is used for the segregation of gram-negative enteric bacteria.
2. It is used in the separation of lactose fermenting from lactose non-fermenting gram-negative bacteria.
3. It is also used for the isolation or segregation of coliforms and intestinal pathogens in water and biological specimens.
4. It is used for the observation of milk and dairy products.
5. British pharmacopoeia has suggested this medium for the subculture and identification of Escherichia coli.

Limitations of MacConkey Agar:

1. The colonial characteristics describe giving probable identification only of the isolated organisms. It is required to subculture and carry out other confirmation tests like for final identification biochemical test.
2. Some strains may be encountered that mature poorly or fail to mature on this medium.
3. Incubation of MacConkey Agar plates under increased CO2 has been noted to reduce the growth and improvement of several strains of Gram-negative bacilli.
4. Some strains of Proteus may mob on this medium.

To summarize, out of so many culture mediums, MacConkey Agar has proved to be a very useful one.  Thus, it is considered the best selective and differentiating agar of non-fastidious gram-negative rods, particularly members of the family Enterobacteriaceae and the genus Pseudomonas. 

Are you wondering where do we obtain Agar from? Keep reading at MBD to find out!

Team MBD

Learn how to interpret MacConkey Agar results- How to Interpret MacConkey’s Agar Bacterial Growth Medium – YouTube