Acetate Utilization Test: Principle, Procedure, Results, Uses

A stool culture grows a non-lactose-fermenting, Gram-negative rod that is oxidase-negative and non-motile. It could be Escherichia coli or it could be Shigella, and the two are so closely related that telling them apart takes a battery of small tests. On acetate agar, one of them grows and turns the slant blue, and the other does not. That single difference is one of the steps that separates a common gut commensal from a dysentery pathogen.

What is the principle of the acetate utilization test?

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.

How do acetate and acetamide tests differ?

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.

Which organisms is the acetate utilization test used for?

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.

What materials are required?

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

How is quality control performed?

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)

How is the acetate utilization test performed?

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 35°C 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.

What results should you expect?

Repeat the tests with equivocal results.

1. Positive test: Growth of the organism and a change in the color of the medium from green to intense blue along the slant. About 84% of E. coli strains are positive for acetate utilization. Most Shigella strains are negative, although certain Shigella flexneri biotypes are an exception and can be positive.
2. Negative: No growth, no color change (i.e., the slant remains green).

How to read the result

Read the slant after incubation. A positive result is growth of the organism together with a color change from green to intense blue along the slant. A negative result is no growth and no color change, so the slant stays green.

The color and the growth go together. The organism grows only if it can use acetate as its sole carbon source, and that growth is what produces the ammonia that raises the pH and turns the indicator blue. This is why a positive result needs both growth and blue color, not color alone.

What is the acetate utilization test used for?

The acetate utilization test is used mainly to help separate Escherichia coli from Shigella. These two are very closely related and can look identical on primary plates, since both are non-lactose-fermenting in the relevant biotypes, oxidase-negative, non-motile, and anaerogenic Gram-negative rods. Distinguishing them matters clinically, because Shigella is a dysentery pathogen while many E. coli are harmless gut flora.

The typical split is:

1. Escherichia coli: positive. About 84% of E. coli strains utilize acetate and turn the slant blue.
2. Shigella: negative. Most Shigella strains cannot use acetate and show no growth or color change.

The test is presumptive, not definitive. It is one step in a battery of biochemical tests, not a stand-alone identification. It is typically used alongside other enteric differentiation tests when the primary plates cannot separate E. coli from Shigella.

What are the limitations of the acetate utilization test?

Not every strain follows the typical pattern, which is why the test is never read in isolation:

1. Some E. coli are acetate-negative or grow slowly, so a negative result does not completely exclude E. coli.
2. Some Shigella can be acetate-positive. Certain Shigella flexneri biotypes are a documented exception, where a substantial proportion of strains utilize acetate. A positive result therefore does not completely exclude Shigella.
3. Other Enterobacteriaceae, including Salmonella, Citrobacter, Klebsiella, Enterobacter, and Serratia, can also grow on acetate, usually within one to seven days. This is why the test is applied to isolates already narrowed down to the E. coli-versus-Shigella decision, not to any Gram-negative rod.

Because of these exceptions, a single acetate result is combined with the rest of the identification, and equivocal results are repeated with a light inoculum.

References and further readings

• Tille, P. M. (2022). Bailey and Scott's Diagnostic Microbiology (15th ed.). Elsevier.
• Leber, A. L. (Ed.). (2016). Clinical Microbiology Procedures Handbook (4th ed.). ASM Press. https://doi.org/10.1128/9781683670438.CMPH