To perform biochemical test of lipid hydrolysis, oxidase test and catalase test

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BACKGROUND

Some bacteria posses the capability to hydrolyze lipids to glycerol and fatty acids and are called lipolytic bacteria. Oxidase test is performed to identify bacteria who can produce cytochrome C oxidase enzyme. Similarly, the third test of this experiment is catalase test which is done to know the presence of enzyme catalase in the bacteria that helps in releasing oxygen from hydrogen peroxide. The basic objective of this test is to identify bacteria on the basis of biochemical test.

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REQUIREMENTS

Materials:   Bacterial colonies

Tributyrin agar

Moist filter paper with the substrate

Tetra methyl-p-phenylenediamine dihydrochloride

3% H₂O₂

Apparatus:  Sterilized petri dishes

  Conical flask

Inoculating loops

Cotton plugs

 Autoclave

       Bunsen burner

 Dispose jar

       Clean slide

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PROCEDURE

Lipid hydrolysis

Prepare Tributyrin agar plates.¹ Then inoculate the test bacteria in a small spot at the centre, inside the laminar airflow. Then the plates are incubated at 37⁰C for about 24 hours. Then after 24 hours if clear zones are formed around inoculating spot, then it is lipid hydrolysis positive and vice versa.

Oxidase test

Take the moist filter paper containing the substrate tetra methyl p-phenylenediamine 1% and make a smear of the test culture with the help of platinum loop in the filter paper.² Then observe for any colour change to deep blue or purple within 10-30 seconds. If colour changes then it is oxidase positive.

Catalase test:

Take a small amount of test bacterial culture with the help of inoculating loop and put in a clean slide.³ Then place a drop of 3% H₂O₂ on it and properly mix. Then if there is evolution of bubbles shows that bacteria is catalase positive and vice versa.

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CONCLUSION

Thus, the above test helps in differentiating bacteria on the basis of their various properties.

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REFERENCES

1. Fisher SK, Agranoff BW. J Neurochemistry, Wiley Online library. 1987
   
2. Hwang KT, Regenstein JM. Journal of Aquatic Food product. 1996.
   
3. Taylor WI, Achanzar D. Applied microbiology, Am Soc Microbial. 1972.