Egg Yolk Agar- Composition, Principle, Preparation, Results, Uses
Egg Yolk Agar is a complex medium that contains the following ingredients and their quantities per liter of distilled water:
- Casein enzymic hydrolysate: 15 g
- Papaic digest of soyabean meal: 5 g
- Yeast extract: 3 g
- Sodium chloride: 5 g
- L-cystine: 0.5 g
- Vitamin K1: 1 mg
- Hemin: 5 mg
- Agar: 16.41 g
- Egg yolk emulsion: 100 ml
The final pH of the medium is adjusted to 7.5±0.2 at 25°C.
Each ingredient has a specific role in the medium:
- Casein enzymic hydrolysate and Papaic digest of soyabean meal provide the essential nutrients along with carbonaceous and nitrogenous substances for the growth of anaerobic bacteria.
- Yeast extract supplies B-complex vitamins and other growth factors.
- Sodium chloride maintains the osmotic equilibrium of the medium.
- L-cystine is an amino acid that also acts as a reducing agent to create an anaerobic environment.
- Vitamin K1 and hemin are essential cofactors for some anaerobic bacteria, especially those belonging to the genus Clostridium.
- Agar is a solidifying agent that gives the medium a firm consistency.
- Egg yolk emulsion is the key ingredient that differentiates Egg Yolk Agar from other media. It contains lecithin, a phospholipid that can be hydrolyzed by lecithinase-producing bacteria, and free fats that can be hydrolyzed by lipase-producing bacteria. It also contains proteins that can be degraded by proteolytic bacteria.
The composition of Egg Yolk Agar makes it suitable for detecting various enzymatic activities of anaerobic bacteria, which can be used for their presumptive identification.
Egg Yolk Agar is a medium that exploits the presence of certain enzymes in some anaerobic bacteria to produce characteristic reactions. These enzymes are lecithinase, lipase, and protease.
Lecithinase is an enzyme that breaks down lecithin, a phospholipid found in egg yolk and animal tissues. Lecithinase-producing bacteria can hydrolyze lecithin to insoluble diglyceride and phosphorylcholine, which results in a white opaque zone of precipitation that spreads beyond the edge of the colony. This zone is also known as the Nagler reaction or the opalescent reaction. Lecithinase is a diffusible enzyme that can affect the entire agar plate.
Lipase is an enzyme that breaks down fats or lipids into glycerol and free fatty acids. Lipase-producing bacteria can hydrolyze the free fats present in the egg yolk medium to form glycerol and free fatty acids. The release of insoluble free fatty acids results in the formation of an iridescent sheen (oil on water) that can be seen when the plate is held at an angle to a light source. This sheen is also known as the Stormy Fermentation reaction. Lipase is not a diffusible enzyme and produces a reaction only on the surface of the agar in the immediate vicinity of the colony.
Protease is an enzyme that breaks down proteins into amino acids or peptides. Protease-producing bacteria can degrade the proteins present in the egg yolk medium to form clear zones in the medium surrounding colony growth. This zone is also known as the proteolytic reaction.
The purpose of Egg Yolk Agar is to detect and differentiate anaerobic bacteria based on their lecithinase, lipase, and protease production. These enzymes are important virulence factors for some anaerobic pathogens, such as Clostridium perfringens, Clostridium difficile, Fusobacterium necrophorum, and Prevotella melaninogenica. By observing the presence or absence of these reactions on Egg Yolk Agar, one can presumptively identify these organisms and confirm their identity with further tests.
Egg Yolk Agar is a ready-to-use medium that can be purchased from commercial suppliers or prepared in the laboratory. The following steps describe how to prepare and use the medium in the laboratory:
- Suspend 50.41 grams of dehydrated Egg Yolk Agar base in 900 ml of distilled water. Mix well and dissolve the medium completely by heating to boiling.
- Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
- Cool the medium to 50-55°C and aseptically add 100 ml of Egg Yolk Emulsion (or 10 ml of sterile egg yolk emulsion per 90 ml of medium). Mix well and pour into sterile Petri plates.
- Prior to inoculation, allow the medium to equilibrate to room temperature. If the medium is not already pre-reduced, it must be reduced by placing it under anaerobic conditions for 18-24 hours.
- Inoculate Egg Yolk Agar with a pure 24-72 hour culture of the test organism. Streak the medium so as to obtain isolated colonies.
- Immediately following inoculation, place the medium in an inverted position (agar side up) in an anaerobic atmosphere and incubate at 35-37°C for 48-72 hours.
- Observe the plates for the appearance of lecithinase and lipase production and proteolytic activity after 48 hours of incubation. Cultures should not be discarded as negative until after 7 days of incubation.
The results of the egg yolk agar test can be interpreted by observing the appearance of the colonies and the surrounding medium after incubation. The following are some of the possible reactions:
- Lecithinase production: This is indicated by a white opaque zone of precipitation that spreads beyond the edge of the colony. This reaction is caused by the breakdown of lecithin to insoluble diglyceride and phosphorylcholine. Lecithinase production is a characteristic feature of Clostridium perfringens, Clostridium bifermentans, Clostridium sordellii, and some strains of Fusobacterium necrophorum.
- Lipase production: This is indicated by an iridescent sheen (oil on water) that can be seen when the plate is held at an angle to a light source. This reaction is caused by the hydrolysis of free fats to glycerol and free fatty acids. Lipase production is a characteristic feature of Clostridium sporogenes, Clostridium difficile, and some strains of Prevotella melaninogenica.
- Proteolysis: This is indicated by the development of clear zones in the medium surrounding colony growth. This reaction is caused by the degradation of proteins to amino acids and peptides. Proteolysis is a characteristic feature of Clostridium histolyticum, Clostridium novyi, and some strains of Fusobacterium nucleatum.
- No reaction: This is indicated by no change in the appearance of the medium or the colonies. This means that the microorganism does not produce lecithinase, lipase, or proteolytic enzymes. Some examples of microorganisms that show no reaction on egg yolk agar are Clostridium tetani, Clostridium botulinum, and Bacteroides fragilis.
The results of the egg yolk agar test should be confirmed by other biochemical and/or serological tests for complete identification of the microorganism. The test should also be compared to an uninoculated control plate, as lecithinase can diffuse throughout the entire agar plate and make interpretation difficult. Some microorganisms may require up to one week to produce a positive lipase reaction, so cultures should not be discarded as negative until after 7 days of incubation.
Egg Yolk Agar is an enriched, non-selective, and differential medium that can be used for various purposes in microbiology. Some of the uses of Egg Yolk Agar are:
- It is recommended for use in the detection of lecithinase and lipase production and proteolytic activity of certain obligate anaerobes. These enzymes are important for the metabolism and virulence of some anaerobic bacteria. Lecithinase hydrolyzes lecithin, a component of cell membranes and egg yolk, to produce a white opaque zone around the colonies. Lipase hydrolyzes fats to produce an iridescent sheen on the surface of the agar. Proteolytic activity is indicated by clear zones around the colonies due to the breakdown of proteins in the medium.
- It is used for the presumptive identification of various Clostridium, Fusobacterium, and Prevotella species. These are gram-positive or gram-negative anaerobic rods that are commonly found in soil, water, and the human gastrointestinal tract. Some of them can cause serious infections such as gas gangrene, tetanus, botulism, and periodontal disease. By observing the lecithinase, lipase, and proteolytic reactions on Egg Yolk Agar, one can differentiate between these genera and other anaerobes.
- It is also used in the Nagler Test for the presumptive identification of Clostridium perfringens. This is a gram-positive anaerobic rod that is responsible for food poisoning and gas gangrene. It produces a potent lecithinase called alpha-toxin that causes tissue necrosis and hemolysis. The Nagler Test involves inoculating two plates of Egg Yolk Agar with a suspected culture. One plate is supplemented with antitoxin that neutralizes the alpha-toxin. The other plate serves as a control. After incubation, a positive result is indicated by a white opaque zone on the control plate but not on the antitoxin plate.
Egg Yolk Agar is a useful medium for studying the biochemical characteristics and pathogenic potential of some anaerobic bacteria. However, it does not provide complete information for the identification of bacterial isolates. Additional biochemical and/or serological tests must be performed on colonies from pure culture for complete identification.
- Egg Yolk Agar is an enriched, non-selective, and differential medium that can detect lecithinase and lipase production and proteolytic activity of certain obligate anaerobes. However, it has some limitations and challenges that should be considered when using it.
- One of the limitations of Egg Yolk Agar is that it does not provide complete information for the identification of bacterial isolates. Additional biochemical and/or serological tests must be performed on colonies from pure culture for complete identification. For example, the Nagler Test for the presumptive identification of Clostridium perfringens requires the use of another medium (Litmus Milk) to confirm the presence of phospholipase C enzyme.
- Another limitation of Egg Yolk Agar is that some microorganisms may require up to one week to produce a positive lipase reaction. This can delay the diagnosis and treatment of infections caused by these organisms. Moreover, some microorganisms may produce weak or variable reactions on Egg Yolk Agar, making the interpretation difficult. For instance, Clostridium sporogenes may produce a weak lecithinase reaction or no reaction at all on Egg Yolk Agar.
- A third limitation of Egg Yolk Agar is that it is not a selective medium and can support the growth of various aerobic and facultative anaerobic bacteria. This can result in overgrowth and contamination of the medium by unwanted organisms. Therefore, it is recommended that an enrichment broth be simultaneously inoculated with the test sample to detect the small number of anaerobic organisms. Furthermore, it is important to use aseptic techniques and anaerobic conditions when preparing and incubating the medium to prevent oxidation and exposure to oxygen.
- A fourth limitation of Egg Yolk Agar is that it can be affected by environmental factors such as temperature, pH, and moisture. These factors can influence the quality and stability of the medium and the reactions of the microorganisms. Therefore, it is essential to follow the manufacturer`s instructions for storage and handling of the medium and to check for any signs of deterioration or contamination before use. It is also advisable to use freshly prepared or pre-reduced medium for optimal results.
In conclusion, Egg Yolk Agar is a useful medium for detecting lecithinase and lipase production and proteolytic activity of certain obligate anaerobes. However, it has some limitations and challenges that should be taken into account when using it. It does not provide complete identification of bacterial isolates, it may require a long incubation time for some microorganisms, it is not selective and can be contaminated by unwanted organisms, and it can be affected by environmental factors. Therefore, it should be used in conjunction with other tests and methods for accurate diagnosis and identification of anaerobic infections.
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