MacConkey agar

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    Introduction

    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 media useful for the isolation of non-fastidious gram-negative rods (mostly members of the family Enterobacteriaceae and the genus Pseudomonas), and differentiation based on their ability to ferment lactose.

    MacConkey agar with lactose fermenting and non fermenting bacterial colonies

    Principle

    To understand how MacConkey agar works, lets first see the role of different ingredients.

    Agar: Solidifying agent

    NaCl: Osmotic balancer

    Peptone: Nitrogen source, also works as a carbon source for bacteria which grow on this media but can not metabolize sugars used.

    Bile salts: Inhibit the growth of gram-positive bacteria by disrupting the membranes. Gram-negative species live in the colon can resist bile salts negative effects.

    Crystal violet: Inhibits growth of gram-positive bacteria.

    Neutral red: A pH indicator acts as a litmus paper. Turns to red/pink when pH drops below 6.8. 

    Lactose: A sugar works as a carbon source. Some bacteria consume the lactose and produce acids (lactose fermenters). When the acids are released into surrounding media, media pH drops. 

    The colour of colonies in MacConkey agar based on their ability to change the pH of media. For more details on pH changes, read EMB Agar.

    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: SalmonellaProteus species, YersiniaPseudomonas aeruginosa and Shigella are an example for this groupThese 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.

    Principle of MacConkey agar

    Uses

    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.

    pH

    The pH of MacConkey agar medium is about 7.1+/- 0.2. Adjust the pH with 1N NaOH and HCl.

    Composition

    IngredientFor 500 ml of mediaFor 1L of media
    Peptone 1.5 gm3 gm
    Pancreatic digest of gelatin8.5 gm17 gm
    Lactose5 gm10 gm
    Bile salts 0.75 gm1.5 gm
    NaCl2.5 gm5 gm
    Crystal violet0.0005 gm (0.5 mg)0.001 gm (1 mg)
    Neutral red0.015 gm (15 mg)0.030 gm (30 mg)
    Agar6.75 gm13.5 gm

    As per the necessity of the user, the fermentable sugar lactose can be replaced in the medium by other sugars. Abilities of gram-negative bacteria to ferment these replacement sugars is analyzed in the same way as is lactose fermentation.

    Reagents

    Along with the reagents mentioned in the table

    • 1N NaOH
    • 1N HCl

    Instruments and other requirements

    • Glass beaker
    • Conical Flask / Erlenmeyer Flask
    • Spatula
    • Measuring Cylinder
    • pH meter
    • Weighing balance
    • Distilled Water
    • Butter Paper
    • Magnetic stirrer and pellet
    • Pipettes and tips
    • Petri plates and/or test tubes

    Procedure

    1. Weigh the ingredients separately with respect to the volume of the media. (Here, we are considering 1L of the media).
    2. Suspend the ingredients (except agar) in a glass beaker containing about 900mL of distilled water.
    3. Dissolve the components in the beaker using a magnetic stirrer. (Heat may be applied to dissolve the medium completely).
    4. Adjust the pH of the medium to the desired value.
    5. Adjust the broth to a final volume of 1L using distilled water
    6. Transfer the broth to conical flask or aliquot into smaller volumes.
    7. Now add agar accordingly with respect to the volume of the media (i.e., 13.5 gm agar for 1L of the media).
    8. Close the mouth of the flask with a cotton plug. Seal it further with paper and rubber band.
    9. Autoclave for 20 min at 15 psi (1.05kg/cm2) on liquid cycle.
    10. However, if antibiotics are to be included, their stock solutions should be filter sterilized prior to addition into the media. These antibiotics must be added after the media is cooled to about 45-50°C.
    11. Mix well and pour into sterile Petri plates or tubes for slants.

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

    Storage

    Store the media plates at 4°C until they are utilized.

    Caution

    Add agar after adjusting the pH of the media. At acidic pH, agar hydrolyzes and results in soft agar plates.

    Limitations

    1. 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.
    2. Some strains may show reduced growth, or they may fail to grow on this medium.
    3. 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.

    Reference

    MacConkey Agar plates protocols.

    Wikipedia.

    Bacterial target sites for biocide action.

    Interactions between Bacteria and Bile Salts in the Gastrointestinal and Hepatobiliary Tracts.

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