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Casein Hydrolysis Test For Bacteria

In this casein hydrolysis test for bacteria post we have briefly explained about casein hydrolysis test principle, objectives, requirements, casein hydrolysis test procedure, uses and limitations.

Casein Hydrolysis Test For Bacteria

The casein hydrolysis test is used to see if specific microorganisms, particularly bacteria and fungi, can produce casein hydrolysis using proteolytic exoenzymes, which is important for the environment.

Casein is a massive polymer of amino acids that accounts for around 85 percent of the protein in milk, as well as the milk’s white colour. Casein is far too big to fit through the cell membrane. To use casein, bacteria release proteolytic exoenzymes (caseinases and peptidases) that hydrolyze the protein in stages to amino acids outside the cell.

After crossing the cell membrane via transport proteins, the amino acids can be utilised by cells for metabolism and growth. Growing an organism on a skim milk agar plate (which provides nutrients and casein) and then examining the plates for hydrolysis is how casein hydrolysis is assessed. Plates with no hydrolysis will be white due to the casein, while those with hydrolysis will have clearing zones around the growth.

Principle

Casein is the main protein found in milk. The milk’s opaque whiteness is due to a substantial polymer and colloidal suspension. Casein is too big to pass through the membrane of a bacterial cell. To use casein, bacteria produce proteolytic exoenzymes (caseinase and peptidase) that break down the protein into amino acids outside of the cell.

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.

Requirements

Culture

1. Bacterial culture

2. Skim milk agar

Media

1. Skim milk powder 28.0gm/L, Tryptone 5.0gm/L, Yeast extract 2.50gm/L, Dextrose (Glucose) 1.0gm/L, Agar 15.0gm/L, Final pH  at 25°C.

Procedure

1. Heavily inoculate a area of the Casein Agar surface from a pure culture of the casein hydrolysis test isolate.

2. Seal plate with cellophane tape, Shrink-Seals, or gas permeable strip. Incubate aerobically at 25-30°C for 24 hours.

3. Evaluate the plate for clearing (hydrolysis) around or directly beneath the colony indicating a positive reaction. Incubate for a full 14 days before being concluding the reaction is negative.

4. A duplicate set of media may be inoculated and incubated at 35-37°C. Occasionally casein hydrolysis test results become positive more rapidly at 35-37°C.

Results

casein hydrolysis test

Positive Test: Clearing is observed around and/or beneath colony growth (Hydrolysis).

Negative Test: No clearing is observed around and/or beneath the inoculum (No Hydrolysis).

A positive reaction is indicating 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.

Quality control

Positive Control

1. Bacillus Subtilis ATCC 6633: Positive Reaction, Clear Zone Surrounding Colonies.

2. Proteus Mirabilis ATCC 2593: Positive Reaction, Clear Zone Surrounding Colonies

3. Pseudomonas Aeruginosa ATCC 27853: Positive Reaction, Clear Zone Surrounding Colonies

4. Serratia marcescens ATCC 8100: Positive Reaction, Clear Zone Surrounding Colonies

Negative Control

1. Enterococcus Faecalis ATCC 29212: Negative Reaction, No Clear Zone Surrounding Colonies

2. Escherichia Coli ATCC 25922: Negative Reaction, No Clear Zone Surrounding Colonies

Further Readings

Reference