There are some bacteria (Clostridium perfringens) can produce enzymes (lipases) that hydrolyze lipids like fats, steroids,oils, lecithins, glycerol etc especially in food. The enzyme lipases is used by these bacteria for the hydrolysis of phospholipid in cell membranes, allowing skin and subcutaneous tissues colonization.This is how lipolytic bacteria or microorganisms invades food by degrate it lipid contents hence accelerating food spoilage.
Food poisoning is one of the most common types of human food borne illness and Clostridium perfringens is one of those bacteria incriminated.Contaminated foods usually involved are cooked meat or poultry products that contain large numbers of viable cells. The production of a heat-labile enterotoxin by this sporulating bacteria cells turn to induces the major symptoms of diarrhea in perfringens poisoning
Egg Yolk Agar Base is a growth medium used for the isolation and identification of Clostridia and other anaerobic lipolytic microorganisms based on their ability to breakdown lecithin and free fatty acid particles present in the egg yolk producing an insoluble opaque precipitate and an iridescent sheen on the surface of the colonies respectively
Principle of Egg Yolk Agar Base
- Proteose peptone provide the essential nutrients along with carbonaceous and nitrogenous substances.
- The phosphates is responsible for buffering the medium
- Sodium chloride maintains the osmotic equilibrium.
- Magnesium sulphate serves as a source of divalent cations along with sulphates.
- Glucose serves as a food source or source of energy.
- The addition of hemin help to enhance the growth of anaerobic organisms.
- Those organisms able to produce the enzyme lecithinase break down lecithin present in the egg yolk emulsion producing an insoluble opaque precipitate around the colonies.
- As for lipase-producing organisms,they break down free fatty acids (in the egg yolk emulsion) forming an iridescent sheen on the surface of the colonies.
There may be a delay in lipase activity, therefore culture plates should not be discarded as negative before incubation for at least a week for an accurate result.
Also proteolytic activity is seen as clear zones around the inoculated colonies .
Composition of Egg Yolk Agar Base
The following ingredients are used for the composition Egg yolk agar base
|Final pH at temperature 25°C||7.6±0.2|
How to Prepare Egg Yolk Agar Base
The preparation of Egg yolk Agar Base depends on each laboratory workload or the numbers of culture plates available. Anyway, note that a standard Petri dish is equivalent to 20ml when completely poured.
- Start by suspending 75.10g in 900 ml distilled water.
- Heat to boiling to dissolve the medium completely.
- Now dispense in 90 ml amounts and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
- Cool to 45-50°C then add 10 ml of sterile egg yolk emulsion per 90 ml of medium.
- Mix well and pour into sterile Petri plates
NB: It is also advisable to incubate a petri dish for 24 hour for quality control in order to avoid wastage of resources and ease early determination of contamination after the preparation.
Appearance of Medium after preparation
The Basal medium appears amber coloured, clear to slightly opalescent gel then Yellow coloured opaque gel forms in Petri plates After the addition of egg yolk emulsion
Type of Inoculums
- Food samples
- water samples
- Wound exudates
Plate reading and Colonies morphology
Incubation should be done at 35-37°C for 48-72 hours when incubated anaerobically and Culture plates should be incubated for up to 7 days before concluding a Negative growth or observation.
|Organism||Lecithinase||Proteolytic activity||Lipase activity|
|Bacteroides fragilis||negative reaction||negative,no clear zone surrounding colonies||negative reaction,no iridescent sheen on the colony surface and medium|
|Clostridium butyricum||negative reaction||positive,clear zone surrounding colonies||negative,no iridescent sheen on the colony surface and medium|
|Clostridium botulinum||negative reaction||positive, clear zone surrounding colonies||negative,no iridescent sheen on the colony surface and medium|
|Clostridium perfringens||positive,opaque zone around the colony||negative,no clear zone surrounding colonies||negative,no iridescent sheen on the colony surface and medium|
|Clostridium sporogenes||negative reaction||positive,clear zone surrounding colonies||positive,iridescent sheen on the colony surface and medium|
- Labbe R., 1989, Clostridium perfringens, In Foodborne Bacterial Pathogens Ed., Doyle M. P., P.191, Marcel Dekker, New York , N.Y.
- Duncan C. L., 1973, A. J. Bacteriol., 113:932
- Atlas R. M., 2004, Handbook of Microbiological Media, 3rd Ed., CRC Press.
- McClung and Toabe, 1947, J. Bacteriol., 53:139
- Murray P. R., Baron J. H., Pfaller M. A., Jorgensen J. H. and Yolken R. H., (Ed.), 2003, Manual of Clinical Microbiology, 8th Ed., American Society for Microbiology, Washington, D.C.