Egg Yolk Agar – Composition, Principle, Preparation, Results, Uses

What is Egg Yolk Agar?

• Egg Yolk Agar is a specialized growth medium used for the isolation and identification of Clostridia and other anaerobic microorganisms. It is a modified version of the original Egg Yolk Agar developed by McClung and Toabe, specifically designed for the purpose of isolating and differentiating organisms based on their ability to produce lecithinase and lipase, as well as their proteolytic activity.
• One of the common causes of human foodborne illness is Clostridium perfringens food poisoning. This type of food poisoning typically occurs when individuals consume cooked meat or poultry products that contain a large number of viable cells of Clostridium perfringens. The major symptoms of diarrhea in perfringens poisoning are induced by a heat-labile enterotoxin produced only by sporulating cells.
• Egg Yolk Agar Base is a slight modification of the original McClung Toabe Agar Base, which is used for the isolation and detection of Clostridium perfringens. The modification involves the addition of hemin to the base. Proteose peptone provides essential nutrients, carbonaceous and nitrogenous substances, while phosphates act as a buffer and sodium chloride helps maintain osmotic equilibrium. Magnesium sulfate serves as a source of divalent cations and sulfates, while glucose provides energy for the organisms. The inclusion of hemin in the medium helps enhance the growth of anaerobic organisms.
• The differentiation of organisms on Egg Yolk Agar is based on their enzymatic activities. Organisms that produce lecithinase break down lecithin present in the egg yolk emulsion, resulting in the formation of an insoluble opaque precipitate around the colonies. Lipase-producing organisms break down free fatty acids in the egg yolk emulsion, leading to the formation of an iridescent sheen on the surface of the colonies. It is important to note that lipase activity may be delayed, so plates should not be discarded as negative before an incubation period of one week. Proteolytic activity is observed as clear zones around the colonies.
• To use Egg Yolk Agar, the test specimen should be directly inoculated onto the media plates. Prior to inoculation, the plates should be reduced by placing them in an anaerobic jar for 18-24 hours. Additionally, an enrichment broth should be inoculated simultaneously with the test sample to detect small numbers of anaerobic organisms. Standard procedures for the isolation of the organism should be followed, and incubation should be carried out for 18-48 hours, with continued incubation for 7 days.
• In summary, Egg Yolk Agar is a specialized growth medium used for the isolation and identification of Clostridia and other anaerobic microorganisms. It allows for the differentiation of organisms based on lecithinase and lipase production, as well as proteolytic activity. The inclusion of hemin in the medium enhances the growth of anaerobic organisms, and careful incubation and observation are necessary to detect the characteristic reactions on the agar plates.

Principle of Egg Yolk Agar

• The principle of Egg Yolk Agar is based on the enzymatic activities of microorganisms and their ability to break down specific components present in the medium. The various ingredients in Egg Yolk Agar serve specific functions in promoting the growth of anaerobic organisms and facilitating the differentiation of these organisms based on their enzymatic activities.
• Casein enzymic hydrolysate and Papaic digest of soybean meal provide essential nutrients, carbonaceous, and nitrogenous substances necessary for the growth of microorganisms. Yeast extract supplies B-complex nutrients, which further support microbial growth. Sodium chloride helps maintain osmotic equilibrium in the medium.
• L-cystine, an amino acid, serves a dual role as both a nutrient and a reducing agent. It provides an essential sulfur source and also helps create anaerobic conditions in the medium, which is favorable for the growth of anaerobic organisms.
• Vitamin K1 and hemin are added to Egg Yolk Agar to enhance the growth of anaerobic organisms. These compounds provide essential factors that support the metabolism and proliferation of anaerobes.
• The differentiation of microorganisms on Egg Yolk Agar is based on two enzymatic activities: lecithinase and lipase. Microorganisms possessing the enzyme lecithinase are capable of breaking down lecithin, a component present in the egg yolk emulsion. This enzymatic breakdown results in the production of insoluble diglyceride and phosphorylcholine. As a visible outcome, a white opaque zone of precipitation is formed around the colonies, extending beyond their edges.
• Microorganisms possessing the enzyme lipase can hydrolyze the free fats present in the medium, leading to the release of glycerol and free fatty acids. The presence of insoluble free fatty acids results in the formation of an iridescent sheen (resembling oil on water) when the agar plate is held at an angle to a light source. It is important to note that the lipase reaction occurs only on the surface of the agar in the immediate vicinity of the colony and is not diffusible like lecithinase.
• Proteolysis, the enzymatic breakdown of proteins, is detected by the development of clear zones in the medium surrounding the colony growth. These clear zones indicate the digestion of proteins by proteolytic microorganisms.
• In summary, the principle of Egg Yolk Agar revolves around the enzymatic activities of microorganisms. Lecithinase and lipase break down specific components in the medium, resulting in the formation of visible reactions such as white opaque zones of precipitation and an iridescent sheen. Additionally, the presence of clear zones indicates proteolysis. These characteristic reactions help differentiate microorganisms based on their enzymatic capabilities when grown on Egg Yolk Agar.

Composition of Egg Yolk Agar

Final pH (at 25°C): 7.5±0.2

Preparation of Egg Yolk Agar

The preparation and method of use of Egg Yolk Agar are as follows:

1. Suspend 50.41 grams of Egg Yolk Agar Base in 900 ml of distilled water.
2. Heat the suspension to boiling in order to completely dissolve the medium.
3. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Allow the medium to cool to a temperature of 50-55°C.
5. Aseptically add 100 ml of Egg Yolk Emulsion to the cooled medium. Alternatively, you can add 10 ml of sterile egg yolk emulsion per 90 ml of medium.
6. Mix the medium thoroughly to ensure even distribution of the added Egg Yolk Emulsion.
7. Pour the prepared medium into sterile Petri plates.
8. Prior to inoculation, allow the medium in the plates to equilibrate to room temperature.
9. If the medium has not been pre-reduced, it must be reduced by placing it under anaerobic conditions for 18-24 hours.
10. Inoculate the Egg Yolk Agar with a pure 24-72 hour culture of the desired microorganism. Streak the medium to obtain isolated colonies.
11. Immediately after inoculation, place the Petri plates in an inverted position (agar side up) in an anaerobic atmosphere.
12. Incubate the plates at 35-37ºC for 48-72 hours.
13. After 48 hours of incubation, observe the plates for the appearance of lecithinase and lipase production, as well as proteolytic activity.
14. Do not discard cultures as negative until after 7 days of incubation, as lipase activity may be delayed and take longer to appear.

By following these steps, the Egg Yolk Agar can be prepared and used effectively for the isolation and differentiation of microorganisms based on their enzymatic activities.

Colony Characterisitcs on Egg Yolk Agar

The result interpretation of Egg Yolk Agar is as follows:

1. Clostridium perfringens:
   • Growth is observed.
   • Lecithinase positive: A white, opaque zone extending from the edge of colonies is seen.
   • Lipase negative: No sheen is observed on the agar surface.
2. Clostridium sporogenes:
   • Growth is observed.
   • Lecithinase negative: No reaction is seen.
   • Lipase positive: An iridescent sheen is observed on the agar surface when the plate is held at an angle to the light source.
3. Bacteroides fragilis:
   • Growth is observed.
   • Lecithinase and lipase negative: No reaction is seen on the agar.
4. Fusobacterium necrophorum:
   • Good and luxuriant growth is observed.
   • Lecithinase negative: No reaction is seen.
   • Lipase positive: An iridescent sheen is observed on the colony surface and medium.
   • No proteolytic activity: No clear zone is observed surrounding the colonies.

These interpretations are based on the specific reactions exhibited by different microorganisms when grown on Egg Yolk Agar. The presence or absence of lecithinase, lipase, and proteolytic activity allows for the differentiation and identification of these microorganisms.

Quality Control of Egg Yolk Agar

The quality control of Egg Yolk Agar involves assessing various parameters and observing the cultural response of specific microorganisms. The following information outlines the quality control measures for Egg Yolk Agar:

1. Appearance: The powder should have a cream to yellow color and should be a homogeneous, free-flowing powder.
2. Gelling: After preparation, the medium should form a firm gel comparable to a 2.5% Agar gel.
3. Color and Clarity of Prepared Medium:
   • Basal medium: The prepared medium should appear medium amber colored, clear to slightly opalescent.
   • After addition of egg yolk emulsion: Yellow-colored opaque gel should form in Petri plates.
4. pH: The pH of a 7.5% w/v aqueous solution should be around 7.6±0.2.
5. Cultural Response: The cultural response of specific microorganisms should be assessed after incubation at 35-37°C for 48-72 hours under anaerobic conditions. It is important to note that plates should be incubated up to 7 days before considering them negative.

The following table presents the expected growth and reactions of specific microorganisms during quality control:

Organism:

• Bacteroides fragilis ATCC 25285: Good-luxuriant growth, negative lecithinase and lipase reactions, and no iridescent sheen on the colony surface and medium. Negative for proteolytic activity.
• Clostridium botulinum ATCC 25763: Good-luxuriant growth, negative lecithinase reaction, negative lipase reaction, and no iridescent sheen on the colony surface and medium. Positive for proteolytic activity, indicated by a clear zone surrounding the colonies.
• Clostridium butyricum ATCC 13732: Good-luxuriant growth, negative lecithinase reaction, negative lipase reaction, and no iridescent sheen on the colony surface and medium. Positive for proteolytic activity, indicated by a clear zone surrounding the colonies.
• Clostridium perfringens ATCC 12924: Good-luxuriant growth, positive lecithinase reaction, indicated by an opaque zone around the colony, negative lipase reaction, and no iridescent sheen on the colony surface and medium. Negative for proteolytic activity.
• Clostridium sporogenes ATCC 11437: Good-luxuriant growth, negative lecithinase reaction, positive lipase reaction, indicated by an iridescent sheen on the colony surface and medium, and positive proteolytic activity, indicated by a clear zone surrounding the colonies.

By observing the growth and reactions of these microorganisms, the quality and performance of Egg Yolk Agar can be assessed, ensuring reliable results in the identification and differentiation of anaerobic organisms.

Uses of Egg Yolk Agar

Egg Yolk Agar has several important uses in microbiology:

1. Detection of Enzymatic Activities: Egg Yolk Agar is an enriched, non-selective, and differential medium commonly used to detect the enzymatic activities of lecithinase and lipase, as well as proteolytic activity, exhibited by certain obligate anaerobic microorganisms. The medium provides specific components that allow for the visualization of these enzymatic reactions.
2. Presumptive Identification of Microorganisms: Egg Yolk Agar is utilized for the presumptive identification of various microorganisms, including Clostridium, Fusobacterium, and Prevotella species. By observing the characteristic reactions on Egg Yolk Agar, such as lecithinase production, lipase production, and proteolytic activity, it is possible to make initial identifications of these microorganisms.
3. Nagler Test: Egg Yolk Agar is also employed in the Nagler Test, a diagnostic test used for the presumptive identification of Clostridium perfringens. In this test, a streak of the suspect organism is made on Egg Yolk Agar, and the appearance of lecithinase activity (indicated by a white, opaque zone extending from the colonies) confirms the presence of Clostridium perfringens. The Nagler Test helps differentiate C. perfringens from other Clostridium species.

Overall, Egg Yolk Agar is a versatile medium used for the detection and identification of specific enzymatic activities in anaerobic microorganisms, as well as for the presumptive identification of certain bacterial species, including Clostridium, Fusobacterium, and Prevotella. It plays a crucial role in microbiological laboratories for differentiating and characterizing these microorganisms based on their biochemical properties.

Limitations of Egg Yolk Agar

There are several limitations associated with the use of Egg Yolk Agar:

1. Incomplete Identification: While Egg Yolk Agar can provide valuable information about the enzymatic activities of microorganisms, it does not offer complete identification. To achieve comprehensive identification, further biochemical, immunological, molecular, or mass spectrometry testing should be performed on colonies from pure culture.
2. Additional Testing Required: Egg Yolk Agar is a differential medium that aids in the presumptive identification of certain bacterial isolates based on their lecithinase and lipase activity. However, additional biochemical and/or serological tests are necessary to obtain a complete identification. These tests are performed on colonies isolated from the medium.
3. Interpretation Challenges: Interpreting the lecithinase test can be challenging. Lecithinase produced by microorganisms can diffuse throughout the entire agar plate, making it difficult to differentiate between positive and negative reactions. It is important to compare the test result to an uninoculated control plate to establish a baseline.
4. Delayed Lipase Reaction: Some microorganisms may require up to one week of incubation to exhibit a positive lipase reaction. It is necessary to extend the incubation period to accurately assess the presence of lipase activity.
5. Enrichment Broth Requirement: To detect small numbers of anaerobic organisms, it is recommended to simultaneously inoculate an enrichment broth along with the test sample. This allows for the potential growth of anaerobic organisms that may not be readily detected on the agar plates alone.

It is important to consider these limitations when utilizing Egg Yolk Agar as a diagnostic tool, and to complement the medium with additional tests and techniques for a more comprehensive identification and characterization of microorganisms.

FAQ

References

• Westblom TU, Madan E, Midkiff BR. Egg yolk emulsion agar, a new medium for the cultivation of Helicobacter pylori. J Clin Microbiol. 1991 Apr;29(4):819-21. doi: 10.1128/jcm.29.4.819-821.1991. PMID: 1890184; PMCID: PMC269878.
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