MacConkey Agar Principles, Composition, Preparation, colony characteristics

The culture media MacConkey agar was developed in 20th century by Alfred Theodore MacConkey. MacConkey agar was the first formulated solid differential media but now known as   a selective and differential culture media commonly used for the isolation of enteric Gram-negative bacteria.Based on the bile salt-neutral red-lactose agar of MacConkey.

The presence of Crystal violet and bile salts incorporated in MacConkey agar help to inhibit the growth of Gram-positive bacteria making it a selective media for fastidious Gram-negative bacteria, such as Neisseria species 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  differentiate lactose fermenting gram negative rods from non lactose fermenting gram negative rods. It is primarily used for detection and isolation of  enterobacteriaceae and Pseudomonas spp.

MacConkey agar is used for the selective isolation and identification of Enterobacteriaceae from samples such as  feces, urine, wastewater and foods.

Principle of selective and differential ability of MacConkey agar

Those Gram-negative enteric bacteria that grow on MacConkey agar are differentiated by their ability to ferment lactose (Sugar). Fermentation of lactose by bacteria leads to  the production of the acid that drops the pH of the media. The drop in pH is indicated by the change of neutral red indictor 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.

As for the selective ability of MacConkey agr,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.

Composition of MacConeky Agar  and the functions

• Enzymatic digest of Gelatin, Casein and Animal tissue: provides nitrogen, vitamins, minerals and amino acids essential for growth.
• Lactose: fermentable carbohydrate providing carbon and energy.
• 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’s below 6.8.As a result of lactose fermentation, the pH of the medium decreases, changing the color of neutral red to pink
• Agar : Solidifying agent

Variation in MacConkey Agar preparation

• Mac Conkey agar:without salt – recommended for urocultures
• Mac Conkey agar:without crystal violet – less selective medium and allows growing of some Gram-positive cocci other than enterococci.
  
• Mac Conkey agar:with lactose replaced by sorbitol – used for EHEC
  (enterohaemorrhagic E. coli) strains isolation which are sorbitol-negative and form colorless colonies. For medium preparation replace 10g lactose with 10g sorbitol.

How to Prepare MacConkey Agar

• According to manufacturer instruction,Suspend the measured amount of powder (See in the agar bottle and generally 50 gram) in 1 L of distilled water and mix thoroughly.
  Heat while constantly agitating and boil for 1 minute to completely dissolve the powder.
  Autoclave at 121°C for 15 minutes.
• Pour plates and allow to cool down

Colonies appearance on MacConkey Agar

• Lactose-fermenting organisms appears  pink to brick red colonies with or without a zone of precipitated bile.
• Non-lactose fermenting organisms appears  colorless or clear colonies

Lactose Fermenters Organisms as seen on MacConkey Agar

• Citrobacter spp.: colonies are light pink after 48 hours.
• Klebsiella spp.: Large Mucoid lactose fermenter colonies appearing pink
• 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 Fermenters  Organisms as seen on MacConkey Agar

• Proteus spp.: may swarm depending on the amount of agar in the medium; characterize by a  foul smell
• Shigella spp.: Shigella sonnei produces flat colonies with jagged edges.
• Yersinia spp.: may be colorless to peach.
• Salmonella spp.: Not a lactose fermenting
• Other organisms showing colorless colonies on MacConkey agar are; Edwardsiella spp, Hafnia spp., Morganella spp., Providencia spp.

As for gram positive bacteria like Staphylococcus aureus,no growth is observed

Source: Medical Laboratory Expo