Acetate Utilization Test: Principle, Procedure, and the Shigella vs E. coli Split

Acetate utilization test is used to determine if an organism can use acetate as the sole source of carbon. Acetate agar containing sodium acetate as the sole carbon source and inorganic ammonium salts as the sole nitrogen source is used to inoculate the organism. Growth is indicative of a positive test for acetate utilization.

When the bacteria metabolize acetate, the ammonium salts are broken down to ammonia, increasing the medium’s pH. The increase in pH turns the bromothymol blue indicator in the medium from green to blue.

Acetate vs acetamide: don't mix them up

These two tests look almost the same and are easy to confuse, but they answer different questions about different organisms.

Memory hook: acet-AMIDE has an amide to chop (enzyme, Pseudomonas); acet-ATE is just a plate to grow on (carbon source, enteric split).

This article covers the acetate test. For the enzyme-based acetamide test used in Pseudomonas identification, see the Acetamide Utilization Test.

Test organism

An acetate utilization test is recommended in differentiating Shigella spp. from Escherichia coli. Test isolates are non-lactose-fermenting, Gram-negative rods that are oxidase negative, non-motile, and anaerogenic, likely to be either E. coli or Shigella.

Materials required

1. Sodium acetate agar slants
2. Sterile inoculating loops
3. Sterile pipette
4. Sterile saline

Quality Control

Perform quality control on new lots of media before using them. Inspect media for evidence of contamination, cracks, dehydration, and bubbles before storage and before use. Discard any blue tubes. Perform the performance testing of the test organisms:

• E. coli ATCC 25922—acetate positive (growth; blue color)
• Shigella flexneri ATCC 12022—acetate negative (no growth or trace of growth)

Procedure of Acetate Utilization Test

1. With a straight inoculating needle, inoculate acetate slant lightly with isolates picked from the center of a well-isolated colony. Do not inoculate from a broth culture, because the growth will be too heavy and there are chances of carryover of media.
2. Place cap loosely on tube.
3. Incubate aerobically at 35oC for up to 5 days
4. Observe a color change from green to blue along the slant.

Figure: Acetate Utilization Test A. Positive B. Negative

Notes:

1. Growth on the slant without an accompanying color change may indicate a positive test. However, if the agar does not turn blue on further incubation, the test should be repeated with less inoculum.
2. To avoid false-positive reactions, use a light inoculum to prevent the carryover of substances from previous media.
3. Do not stab the slant since the test requires an aerobic environment.

Expected results

Repeat the tests with equivocal results.

1. Positive test: Growth of the organism and change in color of the test medium from green to intense blue along the slant. Most (85%) E. coli strains are positive for acetate utilization; most Shigella organisms (some strains of S. flexneri) are negative.
2. Negative: No growth, no color change (i.e., the slant remains green).

References and further readings

• Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
• Andrea J. Linscott, 2016.  *Clinical Microbiology Procedures Handbook,*4th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.