Calcium Caseinate Agar

This medium contains casein, a raw milk source rich in amino acids and nitrogen, which is degraded by the proteolytes to form clearer zones surrounding the colonies in an otherwise turbid medium. Meat peptone and beef extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Calcium hydroxide is the inhibitor. Calcium chloride helps to maintain the pH of the medium. Bacteriological agar is the solidifying agent.

Preparation

Suspend 29.2 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into appropriate containers and sterilize in autoclave at 121 °C for 15 minutes. Pour into Petri dishes, swirling the medium to resuspend the precipitate. If desired, to intensify the turbidity of the medium, 5 to 10 g of skim milk can be added to 1 L of the medium.

Instructions for use

1. Inoculation can be made by streaking the surface of the plate or by using the pour plate method.
2. Incubate at 35 ± 2 °C for 48-72 hours. Casein is degraded by proteolytic organisms and forms clear zones surrounding the colonies.
3. The finished medium is turbid, especially if 5-10 g/L of powdered milk is added.
4. Count the colonies with clearing zones only.
5. Covering the surface of the plate with 5-10% acetic acid can improve differentiation of colonies.

• Frazier, W.C. and Rupp, P. Studies on the proteolytic bacteria of milk. A medium for the direct isolation of caseolytic milk bacteria. J. Bact. 16: 57-63 (1928).