Casein Hydrolysis Test: Principle, Procedure, and Uses

The physiological reactions of microorganisms are crucial in species identification and differentiation. As enzymes catalyze all physiological reactions, detecting the enzyme produced is an essential tool for species identification and differentiation. Therefore, the casein hydrolysis test is one of the important test that helps to identify microorganisms based on detecting exoenzyme (caseinase) activity.

Casein hydrolysis is the degradation of casein protein present in an opaque milk agar due to metabolic activity of organism into soluble end products like amino acids, peptides, etc. This results in a transparent zone around the growth Thus, the test performed to detect the breakdown of casein is known as the casein hydrolysis test. 

Casein, a dominant milk protein, is a macromolecule comprising amino acid subunits linked together by a CO-NH bond. The microorganism cannot uptake casein as it is a large molecule. Therefore, it must undergo step-by-step degradation into peptides and a small chain of amino acids for assimilation. These reactions are catalyzed by proteinases (caseinase) and peptidase enzymes. The proteinase cleaves the peptide (CO-NH ) bond of adjacent amino acids by introducing water molecules to release polypeptides and amino acids. 

The overall reaction;    

Casein→small chain of amino acids, dipeptides, and polypeptides; in the presence of caseinase and water molecules.

Principle of Casein Hydrolysis Test

Microorganisms cannot directly utilize large protein molecules. Some microbiomes can degrade such proteins (casein) by producing extracellular enzymes known as proteinase (caseinase). Milk agar is used to demonstrate such activity in the laboratory. As significant ingredients, the medium comprises skim milk powder, tryptone, yeast extract, dextrose, and agar. Skim milk powder in a medium acts as a casein source and gives the medium opacity and color. The opacity of the medium is due to the deflection of light instead of transmitting it. Similarly, other ingredients like tryptone, yeast extract, and dextrose are the source of amino acid, vitamin B complex, and carbon.

Following inoculation and incubation of the culture agar plate, proteolytic bacteria will form a zone of proteolysis, demonstrating a transparent area surrounding the bacterial growth. The formation of the transparent zone is due to the formation of soluble molecules by the degradation of casein protein, and it represents a positive reaction.

Without proteolytic activity, the whole medium remains opaque even around the growth area, representing a negative reaction.

Objectives of Casein Hydrolysis Test

  1. To determine the capability of specific microorganisms to break down the casein protein.
  2. To differentiate microorganisms based on their capability to produce exoenzymes.

Media used in casein hydrolysis test

Composition of Skim Milk Agar

IngredientsGram per liter
Skim milk powder28.0
Yeast extract2.50

Procedure of Casein Hydrolysis test

The procedure of the test includes the following steps:

  1. Take a skim milk agar plate and inoculate it in a straight line or zigzag line with the help of a sterile inoculating loop.
  2. Incubate the inoculated plate at 25 to 37ºC for 18-24 hours.
  3. Then, observe the presence or absence of a clear zone around the growth of the test organism in the agar plate culture.

Results with Examples

Casein hydrolysis test results
CASEIN HYDROLYSIS TEST RESULTS: This Milk Agar plate was inoculated with Bacillus megaterium (caseinase-positive) above and Micrococcus roseus (caseinase-negative) below.

Positive test result: In the case of the positive result, the clear transparent zone is observed around the growth area or sometimes beneath the growth of the colonies. Examples of microorganism that gives positive test result includes; Lactococcus lactis, Pseudomonas spp, Actinomycetes, Bacillus subtilis, Bacillus cereus, Serratia marcescens, etc.

Negative test result: No clear zone is observed around or beneath the colonies in the negative test. Examples of microorganisms that give negative casein test result include; Escherichia coli, Enterococcus fecalis, etc.

Uses of Casein Hydrolysis Test

The uses of the casein hydrolysis test include;

  1. It helps to identify the organism that produces caseinase.
  2. The casein hydrolysis test helps determine the microorganisms that grow in milk.
  3. It also helps in differentiating Bacillaceae, Enterobacteriaceae, and other families.
  4. The identification of aerobic actinomycetes is easily possible by this test.

Limitations of Casein Hydrolysis Test

The limitation of the casein hydrolysis test includes:

  1. Further, biochemical, immunological, molecular, or mass spectrometry tests are required to identify species.
  2. Some strains might show less growth due to variable nutrient requirements.

Quality control

  • Bacillus subtilis subsp. spizizenni ATCC 6633-positive reaction, clear zone surrounding the colonies.
  • Pseudomonas aeruginosa ATCC 27853-positive reaction, clear zone surrounding the colonies
  • Serratia marcescens ATCC 8100-positive reaction, clear zone surrounding the colonies.
  • Escherichia coli ATCC 25922-negative reaction, no clear zone around the colonies
  • Enterococcus fecalis ATCC 29212-negative reaction, no clear zone around the colonies.


  • Brown A.E. 2009. Benson’s Microbiological Applications: Laboratory Manual in General Microbiology, 11th ed. McGraw-Hill Companies, New York, NY, USA.
  • Wusigate, Li L, Yangchao Luo. Casein and pectin: structures, interactions, and applications.Trends in Food Science and Technology. 2020, DOI:
  • Casein Hydrolysis. American Society For Microbiology. Retrieved on 23rd August 2022, from

Samikshya Acharya

Hello, I am Samikshya Sharma. I have completed my post-graduate study in medical microbiology at the central department of microbiology, TU, Nepal. I hope my articles are helpful to you. Thank you!!

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