LITMUS MILK TEST The litmus milk test is used to differentiate
among microorganisms that enzymatically transform different milk substrates into varied metabolic
end products. Litmus milk broth consists of milk, sugar, lactose, pH indicator litmus,
and milk protein casein. Depending on the types of enzymes produced by different organisms,
a variety of different chemical changes happen in the medium. The pH indicator incorporated
in the medium helps in the detection of the production of acid or alkali and oxidation-reduction
activities of microorganisms. MATERIALS REQUIRED: Tryptic soy broth culture of different microorganisms
Inoculating loop Litmus milk broth
Bunsen burner PROCEDURE: Arrange all the cultures and media in the
Laminar Air Flow. Take tryptic soy broth from the rack containing
24 hour culture. Take an inoculation loop and sterilize it
in the blue flame of the Bunsen burner till red hot and then allow it to cool.
Remove the cotton with the small finger of your right hand and flame the mouth of the
tube. Take a loopful of culture from the tube.
Flame the mouth of the tube again, replace the cotton and return it to the rack.
Take a sterile Litmus milk broth from the rack.
Remove the cotton and flame the mouth of the tube.
Inoculate a loopful of culture in the broth. Flame the mouth of the tube and place it in
the rack. The other two test cultures were inoculated
successively. Incubate the tubes at 37 O C for 24 – 48
hours. After incubation, observe the results. RESULT: Escherichia coli convert the milk sugar lactose
into lactic acid with the help of enzyme ß-galactosidase. The medium turns pink due to curd formation
and gas formation. Pseudomonas aeroginosa hydrolyzes the milk
proteins, primarily casein, into their basic building blocks, namely amino acids. This
is accompanied by the evolution of large quantities of ammonia, resulting in an alkaline pH. The
reaction is known as peptonization where brownish supernatant called whey is formed.
Bacillus cereus also results in the peptonization of the medium with the formation of rennet
curd due to the action of enzyme rennin on casein. This forms paracasein, which in the
presence of calcium ions, is converted to calcium paracaseinate and forms an insoluble
clot. Rennet curd is a soft semi-solid clot that will flow slowly when the tube is tilted.