Feb 28, 2025

Public workspaceTributyrin Assays

DOI
dx.doi.org/10.17504/protocols.io.14egn95epl5d/v1
  • 1University of Birmingham
David Cleary
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DOI: dx.doi.org/10.17504/protocols.io.14egn95epl5d/v1
Protocol Citation: David Cleary 2025. Tributyrin Assays. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn95epl5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 28, 2025
Last Modified: February 28, 2025
Protocol Integer ID: 123575
Abstract
Hydrolysis of lipids is an enzymatic process caused by extracellular lipases. To test production of lipases we use tributyrin, a triglyceride component, as a substrate.
Tributyrin Agar Test
Tributyrin Agar Test
  • Using a sterile inoculating loop, pick up a heavy inoculum from purity culture plate
  • Inoculate the tributyrin agar plate by drawing either a straight line or forming a circular inoculation over the surface
  • Incubate the plates at 37°C for about 24 to 48 hours aerobically for aerobes or facultative, and for about 72 hours anaerobically for anaerobes. 
  • Following incubation, observe for formation of a clear zone of tributyrin hydrolysis around the bacterial colony
  • A positive test is indicated by the formation of a clear transparent zone of hydrolysis (tributyrin hydrolysis) around the bacterial colony (in the line). 
  • A negative test is indicated by no clear zone of hydrolysis around the bacterial growth line (colonies).
Tributyrin Strip Test
Tributyrin Strip Test
  • Using a sterile inoculating loop, pick up a heavy inoculum from purity culture plate
  • Innoculate 25 mL of appropriate growth media and incubate at 37°C o/n
  • Using sterile forceps place one tributyrin strip into the bottom of a well. One per test organism
  • Add 30 μL of o/n growth onto the strip
  • For negative controls add 30 μL of PBS
  • For positive controls add 30 μL of o/n culture of Staphylococcus epidermidis strain VAD8.3
  • Incubate at 37oC and examine for colour change (pink to yellow) which indicates positive production of lipase