Tween 80 hydrolysis test


Tween 80 hydrolysis test is used mainly to differentiate slow–growing Mycobacterium (Scotochromogens) with similar colony appearance, M. gordonae (positive) and M. scrofulaceum (negative). This test is also useful in identifying M. kansasii (positive results within 24 hours).

Those Mycobacterium spp that produce lipase hydrolyse the detergent Tween 80 (polyethylene derivative of sorbitan mono-oleate) with the production of oleic acid and polyoxyethylated sorbitol. In this test, the test organism is incubated in a Tween 80 buffered substrate that contains an indicator neutral red. Tween 80 hydrolysis is detected by a change in color of the indicator from light orange to pink – red due to the production of oleic acid.Materials required: 


Mature colony of the unknown Mycobacterium species isolated on Lowenstein Jensen medium.


Tween 80 phosphate buffered substrate with neutral red indicator.



  • Inoculate 4 ml of sterile Tween 80 phosphate buffered substrate with a loopful of growth of the test organism.
  • Incubate at 35-37 oC for up to 18 days.
  • Examine at 5,10, and 18 days for a change in color of the substrate from light orange to pink-red.



Pink-red substrate (Tween 80 hydrolyzed)                  —— Positive test

No change in colour (No hydrolysis of Tween 80)      —— Negative test



Positive Tween 80 hydrolysis control: Mycobacterium kansasii (usually turns positive within 24 hours.)

Negative Tween 80 hydrolysis control: Use an unlnoculated tube of substrate


The reagent should be stored at 4oC in the dark. Do not store or incubate tubes in the light. The red color of a positive reaction is not due to change in pH, rather is due to hydrolysis of Tween 80, which modifies the optical rotation of light passing through the substrate.







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