MacConkey Agar- Composition, Principle, Preparation, Results, Uses

MacConkey agar is a type of culture medium that is widely used in microbiology to grow and differentiate gram-negative bacteria, especially the members of the family Enterobacteriaceae. It was developed by Alfred Theodore MacConkey in 1905 as the first solid differential medium for the isolation of coliforms from water and fecal samples. Coliforms are bacteria that can ferment lactose and produce acid and gas, such as Escherichia coli, Klebsiella, Enterobacter, and Citrobacter. MacConkey agar can also distinguish between lactose-fermenting and non-lactose-fermenting bacteria based on the color change of the pH indicator neutral red in the medium. Lactose-fermenting bacteria produce red or pink colonies on MacConkey agar, while non-lactose-fermenting bacteria produce colorless or transparent colonies. MacConkey agar is also a selective medium because it contains bile salts and crystal violet, which inhibit the growth of most gram-positive bacteria and some gram-negative bacteria that are not enteric. Therefore, MacConkey agar is useful for isolating and identifying gram-negative enteric pathogens such as Salmonella, Shigella, Proteus, and Pseudomonas aeruginosa from clinical specimens and environmental samples. MacConkey agar can also be modified by replacing lactose with other sugars to test the fermentation abilities of different bacteria. For example, sorbitol-MacConkey agar can be used to detect Escherichia coli O157:H7, which is a non-sorbitol-fermenting strain that causes hemorrhagic colitis and hemolytic uremic syndrome. MacConkey agar is one of the most commonly used media in microbiology laboratories because of its simplicity, reliability, and versatility. It is also recommended by various pharmacopeias and standards for the quality control of food, water, milk, and dairy products. In this article, we will discuss the composition, principle, preparation, results interpretation, uses, and limitations of MacConkey agar in detail.

MacConkey agar is a selective and differential medium that contains the following ingredients and their amounts per liter of distilled water:

• Peptone: 17 g

• Proteose peptone: 3 g

• Lactose monohydrate: 10 g

• Bile salts: 1.5 g

• Sodium chloride: 5 g

• Neutral red: 0.03 g

• Crystal violet: 0.001 g

• Agar: 13.5 g

Each ingredient has a specific role in the medium:

• Peptone and protease peptone provide the essential nutrients, vitamins, and nitrogenous factors required for the growth of microorganisms.

• Lactose monohydrate is the fermentable source of carbohydrates that allows the differentiation of lactose fermenting from lactose non-fermenting gram-negative bacteria.

• Bile salts and crystal violet are inhibitory to most species of gram-positive bacteria, making the medium selective for gram-negative bacteria.

• Sodium chloride maintains the osmotic balance in the medium.

• Neutral red is a pH indicator that turns red when the pH of the medium falls below 6.8, indicating the production of acid from lactose fermentation.

• Agar is a solidifying agent that provides a firm surface for bacterial growth.

MacConkey agar is a differential and selective medium that distinguishes between gram-negative bacteria based on their ability to ferment lactose. It also inhibits the growth of most gram-positive bacteria by the presence of crystal violet and bile salts.

The principle of MacConkey agar is based on the following reactions:

• Lactose fermentation: Bacteria that can ferment lactose produce acid and gas, which lower the pH of the medium and turn the pH indicator neutral red from colorless to red or pink. The acid also precipitates bile salts, forming red or pink colonies surrounded by a cloudy zone in the agar. Examples of lactose fermenters are Escherichia coli, Enterobacter, and Klebsiella.

• Lactose non-fermentation: Bacteria that cannot ferment lactose use peptone as an alternative source of energy, producing ammonia and other alkaline products that raise the pH of the medium and turn the pH indicator neutral red from colorless to yellow or colorless. The alkaline products also prevent the precipitation of bile salts, forming white or colorless colonies with a transparent zone in the agar. Examples of lactose non-fermenters are Salmonella, Shigella, Proteus, and Pseudomonas.

The principle of MacConkey agar can be modified by replacing lactose with other sugars, such as sorbitol, mannitol, or sucrose, to test the fermentation abilities of different gram-negative bacteria. For instance, sorbitol MacConkey agar can be used to differentiate Escherichia coli O157:H7, which is a non-sorbitol fermenter, from other strains of E. coli, which are sorbitol fermenters.

MacConkey agar can be prepared from a dehydrated powder or from individual ingredients. The following steps describe the preparation of MacConkey agar from a dehydrated powder:

• Measure 49.53 grams of dehydrated MacConkey agar powder and add it to 1000 ml of distilled water in a flask or bottle. Mix well to dissolve the powder completely.

• Heat the flask or bottle in a water bath or microwave until the solution boils. Alternatively, you can autoclave the flask or bottle at 121°C for 15 minutes to sterilize and dissolve the medium.

• Cool the solution to 45°C - 50°C. You can use a thermometer to check the temperature or wait until you can hold the flask or bottle comfortably in your hand.

• Pour the solution into sterile Petri dishes. You can use a sterile pipette or a dispenser to transfer the solution. Make sure to avoid bubbles and spills. Each Petri dish should contain about 15-20 ml of medium.

• Allow the medium to solidify at room temperature. Do not move or tilt the Petri dishes until the medium is completely solid.

• Store the prepared MacConkey agar plates in a refrigerator (2°C - 8°C) in an inverted position (lid down) until use. You can also wrap them in plastic bags or foil to prevent dehydration and contamination.

Some tips for preparing MacConkey agar are:

• Do not overheat or overcook the medium, as this may affect its performance and selectivity.

• Do not add any supplements or additives to the medium unless specified by the manufacturer or protocol.

• Do not use tap water or mineral water as they may contain impurities or minerals that may interfere with the medium.

• Do not reuse or reheat the medium once it has been poured into Petri dishes, as this may alter its pH and composition.

• Do not use expired or contaminated dehydrated powder or prepared plates, as this may affect the quality and reliability of the results.

MacConkey agar allows the differentiation of gram-negative bacteria based on their ability to ferment lactose. The medium contains neutral red, a pH indicator that turns red when the pH is below 6.8. Lactose-fermenting bacteria produce acid from lactose, lowering the pH of the medium and turning the colonies and the surrounding agar red or pink. Non-lactose-fermenting bacteria do not produce acid from lactose and remain colorless or white on the medium. Some non-lactose-fermenting bacteria may also produce ammonia from peptone, raising the pH of the medium and turning it transparent or yellow.

Some examples of lactose-fermenting and non-lactose-fermenting bacteria that can grow on MacConkey agar are:

| Lactose-fermenting bacteria | Non-lactose-fermenting bacteria |

| --------------------------- | ------------------------------- |

| Escherichia coli | Salmonella spp. |

| Klebsiella spp. | Shigella spp. |

| Enterobacter spp. | Proteus spp. |

| Citrobacter spp. | Pseudomonas aeruginosa |

Some bacteria may show intermediate or atypical reactions on MacConkey agar, such as:

• Serratia marcescens: It may appear as red colonies with a white center, or as pink colonies with a red center, depending on the strain and incubation conditions.

• Yersinia enterocolitica: It may appear as colorless colonies with a pink "bull`s eye" in the center due to delayed lactose fermentation.

• Edwardsiella tarda: It may appear as colorless colonies with a black center due to hydrogen sulfide production.

To confirm the identity of the isolated bacteria, further biochemical tests, and serological tests are required. MacConkey agar is a presumptive test that only indicates the possible presence of gram-negative enteric bacteria and their lactose fermentation status. It does not provide definitive identification of the species or strain of bacteria.

MacConkey agar is a widely used culture medium for the isolation and differentiation of gram-negative enteric bacteria. It has several applications in microbiology, such as:

• It is used to isolate and identify members of the family Enterobacteriaceae, which are commonly found in the intestinal tract of humans and animals. Some of these bacteria are pathogenic, such as Escherichia coli, Salmonella, Shigella, and Yersinia. MacConkey agar can help to distinguish between lactose-fermenting and non-lactose-fermenting strains of these bacteria based on their colony color and appearance.

• It is used to detect and enumerate coliforms, which are indicators of fecal contamination in water, food, and dairy products. Coliforms are lactose-fermenting bacteria that produce acid and gas from lactose. MacConkey agar can show the presence and number of coliforms by the formation of red or pink colonies surrounded by a turbid zone in the agar.

• It is used to test the ability of gram-negative bacteria to ferment other sugars besides lactose. By replacing lactose with other carbohydrates, such as sorbitol, sucrose, or maltose, MacConkey agar can be modified to test for specific types of bacteria that can utilize these sugars. For example, sorbitol MacConkey agar is used to isolate and identify E. coli O157:H7, which is a non-sorbitol fermenting strain that causes hemorrhagic colitis and hemolytic uremic syndrome.

• It is used for the subculture and identification of E. coli in pharmaceutical products. The British Pharmacopeia has recommended MacConkey agar for this purpose, as E. coli is one of the most common contaminants in drugs and cosmetics. MacConkey agar can help to confirm the presence of E. coli by its characteristic green metallic sheen on the surface of the colonies.

MacConkey agar is a useful and versatile medium for the study of gram-negative enteric bacteria. It can provide valuable information about their morphology, physiology, and pathogenicity. However, it is important to note that MacConkey agar is not a definitive identification medium, and further tests are required to confirm the identity of the isolated organisms.

• MacConkey agar is not a definitive identification medium for gram-negative bacteria. It only provides a presumptive identification based on lactose fermentation and colony morphology. Other tests, such as biochemical tests, are required to confirm the identity of the isolated organisms.

• MacConkey agar may not support the growth of some gram-negative bacteria that have special nutritional requirements or are inhibited by the selective agents in the medium. For example, some strains of Vibrio, Pasteurella, Brucella, and Bordetella may not grow well or at all on MacConkey agar.

• MacConkey agar may not differentiate between some gram-negative bacteria that have similar lactose fermentation patterns. For example, some strains of Klebsiella, Enterobacter, and Citrobacter may produce gas from lactose fermentation, which can cause the colonies to appear pink and mucoid, resembling Escherichia coli. Similarly, some strains of Salmonella and Shigella may produce acid from lactose fermentation, which can cause the colonies to appear red and non-mucoid, resembling other lactose fermenters.

• MacConkey agar may not detect some variants of gram-negative bacteria that have atypical lactose fermentation patterns. For example, some strains of Escherichia coli may be late or non-lactose fermenters, which can cause the colonies to appear white or colorless on MacConkey agar. Conversely, some strains of Yersinia enterocolitica may be lactose fermenters, which can cause the colonies to appear pink or red on MacConkey agar.

• MacConkey agar may be affected by the incubation conditions and the quality of the medium. For example, incubating the plates under increased CO2 may reduce the growth and recovery of some gram-negative bacteria. Also, using expired or contaminated medium may alter the pH or the selective properties of the medium, leading to false results.

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