Egg Yolk Agar Base Selective and Differential Medium for the Isolation of Clostridium perfringens

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Egg Yolk base agar MLTGEEKS

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.
Also read  Motility Indole Urea Principle, Composition, Preparation and Colony Characteristics

Note that

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

Proteose peptone40.000
Disodium phosphate5.000
Monopotassium phosphate1.000
Sodium chloride2.000
Magnesium sulphate0.100
Final pH at temperature 25°C7.6±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

Egg Yolk Agar base
Egg Yolk Agar base

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.

Also read  Diagnostic Enzymology (How to calculate enzyme activity)
OrganismLecithinaseProteolytic activityLipase activity
Bacteroides fragilisnegative reactionnegative,no clear zone surrounding coloniesnegative reaction,no iridescent sheen on the colony surface and medium
Clostridium butyricumnegative reactionpositive,clear zone surrounding coloniesnegative,no iridescent sheen on the colony surface and medium
Clostridium botulinumnegative reactionpositive, clear zone surrounding coloniesnegative,no iridescent sheen on the colony surface and medium
Clostridium perfringenspositive,opaque zone around the colonynegative,no clear zone surrounding coloniesnegative,no iridescent sheen on the colony surface and medium
Clostridium sporogenesnegative reactionpositive,clear zone surrounding coloniespositive,iridescent sheen on the colony surface and medium
Clostridium perfringens on Egg York agar base
Clostridium perfringens on Egg York agar base


  • 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.

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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