Acetate Utilization Test- Principle, Procedure, and Result Interpretation

Objective of Acetate Utilization Test

To differentiate organisms based on the ability to use acetate as the sole source of carbon. Generally used to differentiate Shigella sp. from Escherichia coli.

Principle of Acetate Utilization Test

This test is used to differentiate an organism capable of using acetate as the sole source of carbon. Organisms capable of using sodium acetate grow on the medium, resulting in an alkaline pH, turning the indicator from green to blue.


  • NaC2H3O2 (2 g/l)
  • MgSO4 (0.1 g/l)
  • NaCl (5 g/l)
  • NH4H2PO4 (1 g/l)
  • agar (20 g/l)
  • bromthymol blue indicator (0.8 g/l)
  • pH 6.7.

Procedure of Acetate Utilization Test

  1. With a straight inoculating needle, inoculate acetate slant lightly from an 18- to 24-hour culture. Do not inoculate from a broth culture, because the growth will be too heavy.
  2. Incubate at 35°-37°C for up to 7 days.

Result Interpretation of Acetate Utilization Test

Positive: Medium becomes alkalinized (blue) as a result of the growth and use of acetate.

Negative: No growth or growth with no indicator change to blue.


Some strains of E. coli may use acetate at a very slow rate or not at all, resulting in a false negative reaction in the identification process

Quality Control

Positive: Escherichia coli (ATCC 25922)— growth; blue

Negative: Shigella sonnei (ATCC 25931)— small amount of growth; green

Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier