CASEIN HYDROLYSIS TEST

CASEIN HYDROLYSIS TEST

AIM

            To demonstrate the ability of the bacteria to produce an exoenzyme Caseinase and degrade milk protein Casein.

PRINCIPLE       

            Casein is a macromolecule composed of amino acids linked together by peptide bonds, CO-NH. Some microorganism have the ability to degrade the Casein protein by producing proteolytic exoenzyme, called Proteinase (Caseinase) which breaks the peptide bond CO-NH by introducing water into the molecule, liberating smaller chains of amino acids called peptides, which are later broken down into free amino acids by extracellular or intracellular peptidases. These amino acids are transported through the cell membrane into the intracellular amino acid pool for use in the synthesis of structural and functional cellular proteins. 

            The protein casein is a large polymer of amino acids that make around 85 % of the protein found in milk. Casein is a large protein that is responsible for the white color of milk. Caseinase is an exoenzyme that is produced by some bacteria (Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Pseudomonas sp., Streptomyces sp. and Actinomadura sp.) in order to degrade casein. Casein hydrolysis test is conducted on Skim Milk Agar which is a complex media containing casein, peptone and beef extract. If an organism can produce Caseinase, then there will be a zone of clearing around the bacterial growth. Casein hydrolysis test is very helpful in identifying bacteria that grow in milk, and differentiating among Enterobacteriaceae, Bacillaceae and several other families.

MATERIALS REQUIRED

1. Test bacteria
2. Skim milk agar plate
3. Inoculation loop
4. Incubator

PROCEDURE

a)     Inoculate a Skim Milk agar plate with the bacteria to be tested as a Single line. 

b)     Incubate the inoculated Milk agar plate overnight in an Incubator at 37 °C.

c)     Examine the Skim milk agar plate cultures for the presence or absence of a clear area, or zone of proteolysis (Casein hydrolysis), surrounding the growth of each of the bacterial test organisms.

OBSERVATION AND RESULTS

·     Positive Test – Clearing is observed around and/or beneath colony growth (Casein hydrolysis) (Example – Bacillus subtilis and Pseudomonas aeruginosa).

·       Negative Test – No clearing is observed around and/or beneath the inoculum (Example – Escherichia coli).

(Left – Casein hydrolysis negative; Right – Casein hydrolysis positive)

Figure – 1: Casein hydrolysis test