Lipid Hydrolysis Test: Objective, Principle, Procedure, Uses And Results Interpretation

By Prof Walter Jaoko


  • To determine the ability of the organism to hydrolyse lipid.
  • To identify bacteria capable of producing the exoenzyme lipase.


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, bacterial cells secrete exoenzymes known as lipases outside of the cell that hydrolyze the lipid to fatty acids and glycerol. These bacteria capable of producing exoenzyme lipase are called lipolytic bacteria. The lipids form an emulsion, when dispensed in agar, producing opacity while their hydrolysed end products, glycerol and fatty acids do not form such emulsion with agar, for which they do not produce such opacity; rather produce transparency.

In the lipid hydrolysis test, the test bacteria are grown on agar plates containing tributyrin as the lipid substrate. Tributyrin oil forms an opaque suspension in the agar medium. If the bacteria has the ability to hydrolyse lipids, the bacterial colonies hydrolyse the tributyrin in the medium in the areas surrounding them to soluble glycerol and fatty acids (butyric acid), while the rest of the areas of the plates contain unhydrolysed tributyrin. As a result, transparent clear zones are formed around the colonies, as the hydrolysed products, glycerol and fatty acids, formed around them do not form emulsion with the agar. On the other hand, the rest of the areas of the plates remain opaque, as the unhydrolysed tributyrin in these areas forms an emulsion with the agar.


Reagents And Material Required

  • Petri dishes
  • Conical flask
  • Cotton plugs
  • Inoculating loop
  • Autoclave
  • Bunsen burner
  • Laminar flow chamber
  • Dispose jar
  • Incubator
  • Tributyrin agar
  • Isolated colonies or pure cultures of bacteria.


  1. Two petri dishes are cleaned, covered with craft paper and tied with thread or rubber band.
  2. The ingredients of tributyrin agar medium (excluding tributyrin, which is the lipid component) or its ready-made powder required for 100 ml of the medium is weighed and dissolved in 100 ml of distilled water in a 250 ml conical flask by shaking and swirling.
  3. Its pH is determined using a pH paper or pH meter and adjusted to 7.2 using 0.1N HCI if it is more or using 0.1N NaOH if it is less.
  4. The flask is heated to dissolve the agar in the medium completely.
  5. After cooling to about 90°C, tributyrin is added and emulsified in a Warring blender.
  6. The flask is cotton-plugged, covered with craft paper and tied with thread or rubber band.
  7. The two petri dishes and the conical flask containing tributyrin agar medium are sterilized at 121 °C (15 psi pressure) for 15 minutes in an autoclave.
  8. After sterilization, they are removed from the autoclave and allowed to cool for some time, without allowing the medium to solidify. Cooling of the medium prevents condensation and accumulation of water droplets inside the plates. If the medium has already been prepared and solidified during storage, it has to be liquefied by heating carefully till it melts completely.
  9. To prepare tributyrin agar plates, before the sterilized tributyrin agar medium cools and solidifies, in warm molten condition, it is poured aseptically, preferably inside a laminar flow chamber, into the two sterilized petri dishes (approximately 20 ml each), so that the molten medium covers the bottom of the petri dishes completely.Then, the plates are covered with their lids and allowed to cool, so as to solidify the medium in them. Tributyrin forms an emulsion, when dispensed in agar, producing an opaque medium. Water vapour that may condense on the inner surface of the plates and lids is evaporated by keeping the plates and lids in inverted position in an incubator at 37°C for about 1 hour.
  10. Each plate is marked on the bottom side into four quarters.
  11. “Spot inoculation” of the test bacteria is done aseptically, preferably inside a laminar flow chamber, on the center of each quarter by making a spot (or small smear) of the bacteria with the help of a flame-sterilized loop. The loop is sterilized after each inoculation.
  12.  The inoculated plates are incubated in inverted position, top down, at 37°C for 24 to 48 hours in an incubator till colonies of the bacteria are visible.

Observation And Results Interpretation

  • Lipid hydrolysis positive:Transparent clear zones formed around colonies of bacteria.
  • Lipid hydrolysis negative: Transparent clear zones not formed around colonies of bacteria