Tween 80 hydrolysis test: Principle, Procedure and results

Tween 80 is the trade name of the  detergent polyethylene derivative of sorbitan mono-oleate. Some Mycobacterium species posses an enzyme-lipase, that splits the compound and releases oleic acid  and polyoxyethylated sorbitol.

Positive reactions occur in 1 to 4 days of incubation, and 10 days of incubation are required for confirmation of negative reactions. The color change from orange yellow to red is the positive test. Color change is not due to shift is pH but  is due to hydrolysis of Tween 80, which modifies the optical rotation of light passing through the substrate.


  1. This test is useful in identifying Mycobacterium kansasii (which can produce positive results within 3-6 hours)
  2. To differentiate two scotochromogens with similar appearing colonies, M.gordonae (positive) and M.scrofulaceum (negative)


Mature colony of the unknown Mycobacterium species recovered from clinical material, growth on a Lowenstein Jensen slant.


  1. Supplies
  2. Sterile applicator sticks
  3. Screw-capped tubes (12×100 mm) containing 2.0 mL of deionize water per tube
  4. Media
  5. Lowenstein Jensen slant
  6. Reagents
  7. Tween-80 hydrolysis reagent
  8. 2 drops of Difco reagent added to distilled water to screw capped tubes.
  9. Equipment
  10. Biohazard hood

Quality Control

  1. Positive control: Kansasii (ATCC-12478) incubated for 12 days.
  2. Negative control: Stock culture of intracellulare(ATCC-13950)
  3. Negative reagent: Un-inoculated tube of substrate, incubated for 12 days.


  1. Add two drops of Tween reagent to 1 mL of sterile distilled water in screw-capped tubes
  2. Inoculate a loopful of organism to be tested
  3. Incubate at 35oC in the dark with caps tight
  4. Visually read tubes in 24 hours. If negative, read again at 5 and 10 days. Compare the color of the liquid with that in the control tubes.


A positive result is recorded when the liquid, not the cells, turns from light orange to pink or read. M.kansaii usually turns positive within 24 hours. Read again at 3, 5, and 10-12 days. Record results and discard positives. Discard all tubes at 12 days.

Procedure Notes;

Keep Tween hydrolysis reagent in the dark. Do not store or incubate tubes in the light. The red color of a positive reaction is not due to a pH change, rather to release of neutral red.

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