Lipid Hydrolysis Test
Lipids generally are nonpolar molecules that do not dissolve well in water.  Fats are one type of lipids that are large polymers of fatty acids and glycerol.that are too large to enter the cell membrane.  In order to utilize fats, bacteria cells secrete exoenzymes (lipases) outside of the cell that hydrolyze (digestion by the addition of water) the lipid to fatty acids and glycerol.  The cell can convert glycerol and use it in glycolysis, and the fatty acids can be converted into acetyl-coenzyme A and used in the Citric Acid Cycle.  The cell may also use these precursors to make its own lipids.  Lipid hydrolysis can actually be tasted; it makes food taste rancid.  (However, testing an organism in this manner will not be allowed.)  Lipid hydrolysis (or lipase activity) may be tested by growing an organism on an agar plate providing nutrients and a lipid, and then the plates are checked for hydrolysis of the lipid.  Inclusion of the lipid makes the agar appear opaque.  Plates with hydrolysis will have a clear zone around the growth, whereas those lacking hydrolysis will have no zones of clearing around the growth.  Depending on the species being examined, the lipid may be corn oil, tributyrin, egg yolk, or some other lipid.  (Interesting fact: some lipases may be potent cytolytic virulence factors.  Phospholipases can kill cells by desolving the cell membrane.)

Purpose: to test for lipid hydrolysis by lipases that are secreted outside the cell.  Helpful in identifying bacteria that secrete lipase, including members of Enterobacteriaceae, Fusobacterium, Propionibacterium, Clostridium, Pseudomonas, Mycoplasma, Corynebacterium, and Staphylococcus.

Procedure:
  1. Obtain a lipid agar plate and your culture or cultures of interest.  If testing several cultures, obtain 1 plate/3 cultures, and divide the plate bottom into thirds with a heavy marker.  (Use a heavy marker because lipid agar plates are opaque.)  Appropriately label the plate including what the culture is.
  2. Using the wire loop, aseptically transfer a culture to the plate.  Inoculations are best done by spot streaking a big "W" in an area on the plate.  One does not need to streak for isolated colonies, assuming that the culture is pure.
  3. Remind the instructor to set up a positive and negative control.
  4. Invert the plates and incubate them at 35 degrees C for 1-3 days.  All negative results should be incubated for at least 3 days.  (Enterobacteriaceae only needs a day, but a longer incubation works better for many slower growing species.)
Results:
  1. Obtain and examine the lipid agar plates.  Look for areas of clearing around the bacterial growth, comparing the results to the positive and negative controls.
  2. If an organism produces a clear zone, a zone of lipid hydrolysis, score it as positive.  Otherwise, score it as negative.

References:

Brown A.E. 2009.  Benson's Microbiological Applications: Laboratory Manual in General Microbiology, 11th ed. McGraw-Hill Companies, New York, NY, USA.

Cappuccino J.G. and Sherman N.  2008.  Microbiology: A Laboratory Manual, 8th ed. Pearson Benjamin Cummings, San Francisco, CA, USA.

Harley J.P. and Prescott L.M. 2002. Laboratory Exercises in Microbiology, 5th ed. McGraw-Hill Higher Education, New York, NY, USA.

Leboffe M.J. and Pierce B.E.  2011. Exercises for the Microbiology Laboratory, 4th ed. Morton Publishing Company, Englewood, CO, USA.

Pommerville J.C.  2011.  Alcamo's Laboratory Fundamentals of Microbiology.  Jones & Bartlett Learning, Sudbury, MA, USA.

Zimbro M.J., Power D.A., Miller S.M., Wilson G.E., and Johnson J.A.  2009.  Difco & BBL Manual Manual of Microbiological Culture Media, 2nd ed.  Becton, Dickinson and Company, Sparks, MD, USA.

Instructor Notes:

Depending on the species being examined, different lipids may be used.  Corn oil or tributyrin are recommended.  Spirit Blue Agar may also be used, but if using, make sure that the hydrolysis is complete and not partial.  The students may also need to be able to distinguish the difference between "a clear zone" and "a zone of lightening of the medium," as some species lighten the Spirit Blue Agar but do not digest the lipid.  Proteus mirabilis ATCC14273, Staphylococcus aureus ATCC 6538, and Serratia marcescens ATCC ? are positive for lipase.  Escherichia coli ATCC 25922 and Staphylococcus epidermidis ATCC 14990 are negative for lipase.