MacConkey Agar Composition, Uses, Colony Characteristics

MacConkey agar

• MacConkey Agar is a selective and differentiated culture medium widely used to isolate of Gram-negative enteric bacteria.
• It is selective since it allows for the growth of gram-negative bacteria , and differentiating the bacteria that are gram-negative based on their metabolic process of lactose.
• MacConkey Agar is utilized for the specific isolation and identification for members belonging to the Family Enterobacteriaceae from urine, feces and wastewater as well as from food items.
• The first media technology was invented through Alfred Theodore MacConkey in 20th century.
• The most important elements that make up the MacConkey medium are the crystal violet colorant, salts of bile lactose, and neutral Red (pH indicator) All of which have an exclusive characteristic.
• The dye crystal violet and the Bile salts block the growth of Gram-positive bacteria as well as the fastidious Gram-negative bacteria (such as Neisseria and Pasteurella) thus making it suitable in favor of the proliferation of Gram-negative bacteria.
• Because Gram-negative enteric bacteria have an outer membrane with bile resistance that is immune to the bile salts.
• The lactose in the medium is utilized by the bacteria to produce lactic acid, which reduces the pH of the agar, as well as turning indicators (neutral the color is red) pink, which differentiates lactose fermenters from non-lactose ones.

Principle of MacConkey Agar

MacConkey Agar is the earliest differentiated and selective medium used to cultivate coliform bacteria. In the following years, MacConkey Agar and Broth have been suggested for use in microbiological testing of foodstuffs as well as for direct plating/inoculation of water samples to determine the coliform count. The medium is also endorsed as a part of The Standard Methods for the Examination of Milk and Dairy Products. British Pharmacopoeia has recommended this medium for subculture and detection of Escherichia coli. It is also listed by the name of Agar Medium H. It is also suggested by and in line to the harmonized method USP/BP/EP/JP.

Pancreatic digests of gelatin as well as peptones (meat and casein) supply the necessary nutrients, vitamins, and nitrogenous elements required for the development of microorganisms. Lactose monohydrate can be fermented as a sources of carbohydrates. The selective nature of this medium can be attributed the crystal violets and the bile sodium that are inhibiting to the majority of species of Gram-positive bacteria. Salts of sodium chloride help maintain the equilibrium of the osmotic environment within the medium.

Following enrichment Escherichia bacteria by enriching Escherichia coli MacConkey Broth, it is subcultured onto MacConkey Agar. Gramnegative bacteria generally thrive on the medium , and are distinguished by their ability to produce lactose. The strains that ferment lactose appear pink or red, and could be enclosed by a layer of acid precipitated the bile.

The red hue result from the production of acid by lactose, absorbtion of neutral red, and the subsequent change in colour of the dye when the pH of the medium is below 6.8. Non-fermenting lactose strains like Shigella as well as Salmonella are transparent and colorless and generally do not alter the how the medium appears. Yersinia enterocolitica can appear as tiny, non-lactose fermenting colonies following incubation at temperatures of room temperatures.

MacConkey Agar Composition

Ingredients	Amount
Peptone (Pancreatic digest of gelatin)	17 gm
Proteose peptone (meat and casein)	3 gm
Lactose monohydrate	10 gm
Bile salts	1.5 gm
Sodium chloride	5 gm
Neutral red	0.03 gm
Crystal Violet	0.001 gm
Agar	13.5 gm

Preparation MacConkey Agar

1. Submerge 49.53 grams dehydrated media in 1000 ml pure or distilled water.
2. Bring the temperature to a boil until the medium is completely dissolving.
3. Sterilize using autoclaving at 15lbs in pressure (121degC) over 15 mins i.e. the cycle has been validated.
4. Beware of overheating. Cool to 45-50°C.
5. Mix thoroughly before pouring into sterilized Petri plates.
6. Surface of medium needs to be dry upon inoculation.

Morphology of bacterial colony on MacConkey Agar

Organism	Type	Differentiating Colonial characteristics
Escherichia coli	Lactose fermenter	Flat, dry, pink, non-mucoid colonies with a surrounding darker pink area of precipitated bile salts.
Klebsiella spp	Lactose fermenter	Colonies typically appear large, mucoid, and pink, with pink- red pigment usually diffusing into the surrounding agar
Citrobacter spp	Late lactose fermenter	Appear as non-lactose fermenter (NLF) up to 24 hours; however, after 48 hours colonies are light pink.
Enterobacter spp	Lactose fermenter	Pink, mucoid colonies but smaller than Klebsiella spp.
Serratia spp	Late lactose fermenter	S. marcescens may be red-pigmented, especially if the plate is left at 25°C
Proteus spp	Non-Lactose Fermenter	Pale colonies with swarming, characteristic foul smell.
Shigella spp	Non-Lactose Fermenter except S.sonnei which is a late lactose fermenter	Pale colonies,1-2 mm, flat colonies with jagged edges
Providencia spp	Non-Lactose Fermenter	Colonies are colorless, flat, 2-3mm in diameter and do not swarm.
Salmonella spp	Non-Lactose Fermenter	Colorless colonies,convex,2-3 mm with serrated margin
Pseudomonas spp	Non-Lactose Fermenter	Colorless, flat, smooth colonies, 2-3mm in diameter with greenish to brownish pigmentation.
Yersinia spp	Non-Lactose Fermenter	Colonies may be colorless to peach
Gram positive bacteria		No growth

In MacConkey agar without Crystal violet and bile salts

Staphylococcus spp	Lactose fermenter	Small pink colonies, 1-2mm in diameter, opaque
Enterococcus spp	Lactose fermenter	Dark pink to red, very minute, translucent colonies

Modifications of MacConkey Agar

• MacConkey Agar without Crystal Violet: MacConkey Agar without Crystal Violet is a differentiating medium, but it is not as specific than MacConkey Agar. The absence of crystal violet facilitates the expansion in Staphylococcus and enterococci. Staphylococci form tiny red and pale pink colonies while enterococci form compact tiny red colonies, either under or on in the media’s surface. The medium also serves to distinguish Mycobacterium fortuitum, and M. Chelonae from the other mycobacteria growing rapidly.
• MacConkey Agar, CS (“Controlled Swarming”):  MacConkey agar without crystal violet or salt is used to stop the spread from Proteus spp.
• Sorbitol MacConkey Agar: Sorbitol MacConkey Agar an alteration of MacConkey agar. It contains lactose in place of sorbitol to create fermentable sugar. The ingredients of Sorbitol MacConkey the agar comprise peptone, bile salts, sodium chloride Crystal violet, neutral red and Agar. E.coli (VTEC) 0157 non-sorbitol fermenting and produces colorless colonies. The majority of E.coli strains and enterobacteria ferment with sorbitol. Sorbitol-fermenting organisms produce pink colonies.

Quality control of Sorbitol MacConkey agar: 

• Escherichia Coli ATCC(r) 25922 Excellent growth The pink colonies are positive for sorbitol.
• Escherichia Coli ATCC(r) 35150 Excellent growth colony colorless and positive for sorbito.

Uses of MacConkey Agar

• MacConkey Agar is utilized for the isolation of enteric gram-negative bacteria.
• It is utilized in the process of separating lactose fermentation from non-fermenting gram-negative bacilli.
• It is employed for microbiological analysis of food products and to direct inoculation or plating of water samples to determine the coliform count.
• It is used to conduct the analysis of dairy and milk products.
• British Pharmacopoeia has recommended this medium to study the subculture and the identification of Escherichia bacteria.

Limitations of MacConkey Agar

• The colonial characteristics offer the presumptive identification of isolated organisms. It is essential to subculture the colony and conduct other confirmation tests , such as biochemical tests to determine the final identity.
• There are some strains that do not grow well or do not grow well in this environment.
• The incubation on MacConkey Agar plates with an increase in CO2 has been found to slow down growth and improve recovery of various Gram-negative strains of bacilli.
• Certain forms of Proteus might swarm over this medium.

References

• http://www.himedialabs.com/TD/M081B.pdf
• http://www.himedialabs.com/TD/M008S.pdf
• https://www.teknova.com/category-s/504.htm
• http://www.bd.com/resource.aspx?IDX=8978
• https://microbeonline.com/macconkey-agar-mac-composition-preparation-uses-and-colony-characteristics/