Casein Hydrolysis Test

Objective:

 To demonstrate the ability of certain microorganisms to produce extracellular enzymes,    capable of degrading the milk protein casein.

Principle:

           

            Casein is the principle protein of the milk. It is the large polymer and colloidal suspension which gives opaque whiteness to the milk.   Casein is too large to enter into the bacterial cell membrane. In order to utilize the casein, bacterial cells secrete proteolytic exoenzymes (caseinase and peptidase) outside of the cell that break down the protein into amino acids.  The amino acids can then be used by cells after crossing the cell membrane via transport proteins. Casein hydrolysis is tested by growing an organism on a skim milk agar plate (providing nutrients and the casein) and then checking the plates for hydrolysis.  Plates without any hydrolysis will be white from the casein, whereas those exhibiting hydrolysis will have zones of clearing around the growth.

Materials required:
 - Bacterial culture (Bacillus sp.,)
- Skim milk agar

Procedure:
1.      Inoculate the bacterial cultures on skim milk agar medium by streaking. 
2.      The plates are incubating at 37° C for 24 hrs. 
3.      Following incubation, observe the zone of hydrolysis around the line of growth.

Observation:
-   Zone of clearance around the colonies of Bacillus sp.,

      

Result : 
-  Bacillus sp., shows positive result in casein hydrolysis  

       

Interpretation:

            A positive reaction is indicate by the zone of hydrolysis around the colonies of Bacillus sp. This is due to breakdown of casein by caseinase and other enzymes. In negative reaction indicates absence of enzymes showing no zone of hydrolyzing.

Casein hydrolyzing organisms:

Bacillus subtilis., Pseudomonas sp.,

Skim Milk Agar Composition:

Skim milk powder    100 g

Peptone    5g

Agar       15 g

Distilled water 1000 ml