MacConkey Agar (MAC): Composition, Preparation, Uses and colony Characteristics

By Prof Jeremiah Seni

What Is MacConkey Agar?

MacConkey agar (MAC) was developed as the first solid differential media in the 20th century by a bacteriologist, Alfred Theodore MacConkey. While working for the Royal Commission on Sewage Disposal, His role was to inspect drinking water sources for the presence of Gram-negative enteric organisms. Which usually inhabit the gastrointestinal tract of human and other animals as well. Their presence is an indicator of faecal contamination, which can signify the presence of potentially pathogenic bacteria. 

MacConkey agar is a selective and differential culture medium for bacteria. It is designed to selectively isolate Gram-negative and enteric (normally found in the intestinal tract) bacilli and differentiate them based on lactose fermentation. Lactose fermenters turn red or pink on McConkey agar, and nonfermenters do not change color. The media inhibits growth of Gram-positive organisms with crystal violet and bile salts, allowing for the selection and isolation of gram-negative bacteria. The media detects lactose fermentation by enteric bacteria with the pH indicator neutral red.

Composition Of MacConkey Agar Medium

This medium is routinely used in Microbiology laboratory consists of various components and each component plays an important role which is described below in detail…..

Principle

MacConkey Agar Medium (MAC), the selective medium for Gram-negative bacteria, the differential media that differentiate bacteria as Lactose and Non-Lactose fermentors and the Indicator medium by showing two colors of colonies as per the pH – the acidic one as Pink colored and Neutral one as colorless routinely used in Microbiology laboratory consists of various components and each component plays an important role which is described below…..

Peptone – It is a semi-digested protein which is soluble in water and easily metabolized by the bacterial cell, provides the rich source of protein to the bacterial cell of the rapid growth.

Pancreatic digest of gelatin – Similar to Peptone, the Pancreatic Digest of Gelatin is a rich source of Nutrients, Vitamins, and Nitrogenous substances that support the rapid growth of the bacterial cell.

Lactose – The Lactose present in the MacConkey Agar Medium (MAC) provides a rich source of carbohydrate for the rapid growth of the Bacterial cell and is the basis of making the MAC a differential medium, categorizing the bacteria into Lactose and Non-Lactose fermentors.

Bile salt – The Bile salts present in MacConkey Agar medium (MAC) is the basis for making MAC a Selective media. It inhibits the growth of most of the Gram-Positive bacteria and the Gram-Negative remains unaffected.

Sodium Chloride – It maintains the osmotic pressure in the broth medium so that the movement of molecules takes place in and out of the bacterial cell. It must be present in right proportion otherwise it will lead to the lysis of the bacterial cell.

Crystal Violet – Similar in action to the Bile salts, the Crystal violet present in MacConkey Agar Medium (MAC) makes the medium selective for Gram-Negative Bacteria by inhibiting the growth of Gram-Positive Bacteria.

Neutral Red – Here comes the main ingredient that makes the MacConkey Agar Medium an Indicator medium. The Neutral Red is a Dye present in the MAC is the pH-dependent Dye that changes its color as Pink to Red in the Acidic medium. If the Bacterial cell is capable of fermenting the Lactose present in MAC, the pH of the medium changes to Acidic and hence the colonies appear as Pink colored.

Agar-Agar – It is often called as Agar, is a complex polysaccharide, a carbohydrate consisting of 3, 6-anhydro-L-galactose and D-galactopyranose, free of nitrogen, produced from various red-purple algae belonging to Gelidium, Gracilaria, Gigastina etc. It liquefies on heating to 96 °C and hardens into a jelly on cooling 40-45 °C. It basically solidifies the medium and commonly used in the microbiology laboratory.

The final pH of the solutions is adjusted to 6.9 – 7.3 preferably the 7.1 at 25 °C. The above-mentioned components can be modified by adding the various substances in a variety of ways as per the results required so that the rapid and satisfactory growth of the bacterial cell takes place.

Strong lactose fermenters

 Escherichia coli is a typical example for this group. These bacteria change the pH of surrounding media drastically results in pink colour colonies and pink halo. The pink colour of the colonies is due to the change in the colour of neutral red under acidic environment. The dye is also taken up by the bacterial cells results in the pink colonies. 

Since these species can produce strong acids, the acids released into the media could reduce the pH of the areas beyond the colony. The pink halo is a narrow white region at the junction of pink colour (produced by colonies) and the pH unaffected area. The halo is the product of bile salt precipitation due to high acidity. 

Weak lactose fermenters

Serratia and Enterobacter aerogenes are an example for this group. These species change the pH other media to acidic but not as much as the strong fermenters do. The drop in pH is just enough to change the colour of the dye. These bacteria also take up the dye, give the pink appetence to colonies. However, they do not drop the pH of the media to the extent of bile salts precipitation. 

Colonies that do not change the pH of media 

Salmonella, Proteus species, Yersinia, Pseudomonas aeruginosa and Shigella are an example for this group. These bacteria do not ferment lactose; hence neutral red does not change to pink. Some of these bacteria can change the pH to alkali by protein deamination. Increasing the pH of the media may result in a change in the colour of media to yellow as neutral red turns yellow at alkaline pH.

Experiment

Reagents And Material Required

• Sterile Conical Flask / Erlenmeyer Flask
• Spatula
• Peptone (Meat & Casein)
• Pancreatic Digest of Gelatin
• Lactose Monohydrate
• Bile Salts
• Sodium Chloride
• Crystal violet powder
• Neutral Red powder
• Agar-Agar powder
• Measuring Cylinder
• 1N HCl
• 1N NaOH
• pH Strip
• Weighing Scale
• Distilled Wat

Procedure

1. Weigh the quantity of Peptone (3.0g), Lactose (10.0g), the Pancreatic digest of gelatin (17.0g), Bile salts (1.5g), Sodium chloride (5.0g), Crystal violet (0.001g), Neutral red (0.030) and Agar (13.5g).
2. Take a clean and dry Conical Flask/ Erlenmeyer flask.
3. Pour 500 ml of distilled water to the flask and add the weighed quantity of Peptone, Lactose, Pancreatic digest of gelatin, Bile salts, Sodium chloride, Crystal violet and Neutral red. Mix well the content by swirling the flask.
4. Now add the weighed quantity of Agar-Agar to the above solution.
5. Mix well the content and Heat it with continuous agitation to dissolve the constituents.
6. Now add more distilled water to the medium and make the volume 1000 ml.
7. Check the pH of the solution using pH strip, it should be 7.1 ± 0.2. If required, adjust the pH by adding either 1N HCl (acid) or 1N NaOH (base) as per the case.
8. Mix well the content and apply the Non-absorbent cotton plug to the flask.
9. Autoclave the content at 121 °C and 15 psi pressure for 15 minutes.
10. Allow the content to cool down to 40-45 °C and pour in the empty media plates under a strict aseptic atmosphere (preferably in Laminar Air Flow) and allowed it to cool at room temperature.
11. Use the prepared media plates to inoculate the specimen to be cultured and then place in the incubator at optimum temperature.

Alternatively, the commercially available MacConkey agar media powders can be used. Weigh the mixture of content as prescribed by the manufacturer.

Modifications In MacConkey Broth Medium

The MacConkey Broth Medium can be modified in no. of ways as per the requirements of the organisms and to prevent the growth of contaminants and to produce a controlled growth of certain microbes etc. 

• MacConkey Broth Medium lacking Bile Salt:It is used whenever there is need to prevent the swarming of Proteus species.
• Addition of Bromo cresol purple dye instead of Neutral red (MacConkey Broth Purple Medium): The Bromo cresol purple dye is also an excellent indicator which turns Yellow in the Acidic pH, indicating the production of acid and ultimately the fermentation of sugar in the medium by the test organism.
• The Sorbitol MacConkey Broth Medium: It contains Sorbitol as a source of carbohydrate instead of lactose, specifically used to differentiate Pathogenic strains of E.coli from non-pathogenic strains but commonly the Sorbitol MacConkey Agar medium is used for this purpose.
• MacConkey Broth Medium without Crystal Violet: is an excellent modification which is used in laboratories, is a good differential medium due to the presence of crystal violet make it more selective and inhibits the growth of Staphylococcus species and Enterococcus species in the medium. The final pH of the content is same as 7.1 ± 0.2 at 25ºC.

Precautions To Be Taken Into Account When Preparing MacConkey Medium

• Carefully measure the quantity of Peptone, Lactose, Bile salts, Sodium chloride, and Neutral red as per the quantity of medium to be prepared.
• Maintain the pH of the medium carefully. A single extra drop can change the pH of the entire solution.
• Store the Medium at low temperature in dust and contamination free environment for later use.
• Acid and Alkali are corrosive to the skin, handle with care.

Results Interpretation 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.The red color is due to production of acid from lactose, absorption of neutral red and a subsequent color change of dye when the pH of the medium falls below 6.8.
• 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 and typically do not alter appearance of the medium e.g., species of Proteus, Edwardsiella, Salmonella, and Shigella.

Uses Of MacConkey Agar

1. MacConkey agar is routinely used as selective media for the isolation of non-fastidious gram-negative bacteria from wounds, stool, urine and blood samples.
2. It is used as a differential media and an indicator media to distinguish Gram-negative bacteria that can ferment the lactose from those that cannot by using neutral red pH indicator.
3. It is used for testing the quality of water and dairy products by isolation and analyzing the count of coliforms and intestinal pathogens.
   

Limitations of MacConkey Agar

1. Some strains of Proteus may swarm on this medium.
2. Only presumptive identification is possible by observing colony morphology. However, for the final identification, they have to be subcultured, and confirmation tests should be done.
3. Some strains may show reduced growth, or they may fail to grow on this medium.
4. Increased levels of CO₂ during incubation of MacConkey Agar plates has been reported to reduce growth and recovery of a number of strains of Gram-negative bacilli.