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29: Lipid Hydrolysis

Objectives

  • Identify the presence of the enzyme lipase

The enzyme lipase is excreted out of the cells (an exoenzyme) into the surrounding media, catalyzing the breakdown of tributyrin, a vegetable oil, into fatty acids which can then be taken up by the organism. This reaction causes the agar, normally a light blue opaque color to clear around the growth area.

MATERIALS NEEDED

  • 1 tributyrin agar plate with methylene blue dye per unknown

THE PROCEDURE

  1. Run this test using your unknown bacterium.
  2. Inoculate the organisms on the plate either a straight line or a zig-zag.
  3. Incubate at 25º C or 37º C.
  4. Record the results of your bacterial unknown in your journal.

INTERPRETATION

 

 

Tributyrin oil forms an opaque suspension in the agar. When an organism produces lipase and breaks down the tributyrin, a clear halo surrounds the areas where the lipase producing organism has grown. Hold the plate up to the light to see the zones well.

Record positive reactions as weak + or strong +. There is already an indicator, methylene blue, incorporated into the agar. A zone of clearing around the growth area identifies the presence of the enzyme lipase.

QUESTIONS

  1. What is the substrate in this medium?
  2. Name the indicator in this medium.

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