Egg Yolk Agar modified is based upon the original Egg Yolk Agar formula developed by McClung & Toabe to isolate and differentiate organisms based in Lecithinase, lipase production, and proteolytic activities.
Principle of Egg Yolk Agar
Papaic digest of soyabean meals and casein enzymic hydrolysate provide essential nutrients. Yeast extract provides B-complex nutrients. Sodium chloride helps maintain the osmotic balance. L-cystine, an amino acid that also acts as a reducer, is another example. Vitamin K1 (hemin) and vitamin K1 (hemin) help to increase the growth of anaerobic organisms.
The enzyme lecithinase is a microorganism that breaks down lecithin into insoluble diglyceride, phosphorylcholine and glycerol. This results in a white opaque area of precipitation that extends beyond the colony’s edge. The enzyme lipase is able to hydrolyze free fats in the medium and form glycerol or free fatty acids.
The insoluble free fat acids are released, resulting in an iridescent sheen (oil-on-water) that can be seen when the plate has been held at an angle to the light source. Lipase is more stable than lecithinase and reacts only on the surface or colony. Clear zones are seen in the colony’s medium.
Composition of Egg Yolk Agar
|Casein enzymic hydrolysate||15.000|
|Papaic digest of soybean meal||5.000|
Final pH (at 25°C): 7.5±0.2
Preparation and Method of Use of Egg Yolk Agar
- Take 50.41 grams and mix it with 900 ml of distilled water.
- To dissolve the medium completely, heat to boiling
- For 15 minutes, sterilize by using an autoclave at 15 lbs pressure (121degC).
- Cool the medium to 50-55degC, then add 100ml Egg Yolk Emulsion or 10ml of sterile yolk emulsion for every 90ml.
- Mix everything together and pour onto sterilized Petri dishes.
- Allow medium to reach room temperature before inoculation.
- If the medium has not been pre-reduced it must be reduced by placing in anaerobic conditions for between 18-24 hours.
- Modified egg yolk agar. To obtain isolated colonies, you must soak the medium.
- Place the medium inverted (agar side down) immediately after inoculation. Incubate at 35 to 37oC For 48-72 hours.
- After 48 hours, observe plates to see if there is any lecithinase or lipase production. After 7 days, cultures should not be considered negative.
Result Interpretation of Egg Yolk Agar
|Clostridium perfringens||Growth; lecithinase positive; white, opaque zone extending from the edge of colonies, lipase negative; no sheen|
|Clostridium sporogenes||Growth; lecithinase negative; lipase positive; an iridescent sheen on agar surface when the plate is held at an angle to the light source|
|Bacteroides fragilis||Growth; lecithinase and lipase negative; no reaction on agar|
|Fusobacterium necrophorum||Good-luxuriant growth; negative reaction for lecithinase; positive for lipase reaction iridescent sheen on the colony surface and medium; no proteolytic activity no clear zone surrounding colonies|
Uses of Egg Yolk Agar
- It is an enriched, differential, non-selective medium that can be used to detect lecithinase, lipase, and the proteolytic activity for certain obligate anaerobes.
- It is used to presumptively identify various Clostridium, Fusobacterium and Prevotella.
- It’s also used in the Nagler test to presumptive identify Clostridium perfringens.
Limitations of Egg Yolk Agar
- For complete identification, it is recommended that colonies grown from pure culture be subject to biochemical, immunological and molecular testing.
- The medium doesn’t provide enough information to identify bacterial isolates. For complete identification, additional biochemical or serological tests must also be done on colonies grown in pure culture.
- Negative lecithinase tests should be compared with uninoculated control plates, as lecithinase may diffuse across the entire plate making interpretation difficult.
- Some microorganisms can take up to a week to produce a positive reaction to lipase.
- To detect small numbers of anaerobic bacteria, it is important to inoculate the enrichment broth with the test sample.