Acetamide Utilization Test- Principle, Procedure, and Result Interpretation

What is Acetamide Utilization Test?

The Acetamide utilization test is one of the many biochemical tests performed for the identification of aerobic organisms.

This test is based on the utilization of acetamide by the organism via the process of deamidation. The deamidation process occurs in the presence of acylamidase enzyme. An Acetamide utilization test is performed for the distinction between the fermentative group of organisms and the oxidative group of organisms.

This has been commonly used for the differentiation or identification of Gram-negative, non-fermentative group of bacteria like Pseudomonas aeruginosa, on the basis of their ability to utilize acetamide.

A medium with acetamide as the sole source of carbon is used to observe the utilization of acetamide by the organism grown on the medium. The change in color of the medium after growth gives the result of the test.

A similar test to the acetamide utilization test is the citrate utilization test where the organism is identified on the basis of its ability to utilize citrate as a sole source of carbon. This is used for the identification of organisms belonging to the Enterobacteriaceae family.

Objective of Acetamide Utilization Test

To differentiate microorganisms based on the ability to use acetamide as the sole source of carbon.

Principle of Acetamide Utilization Test

Bacteria capable of growth on this medium produce the enzyme acylamidase, which deaminates acetamide to release ammonia. The production of ammonia results in an alkaline pH, causing the medium to change color from green to royal blue.

Acetamide agar is used to determine the ability of an organism to utilize acetamide as a sole source of carbon by deamidation. The medium consists of acetamide, which is the sole carbon source, and inorganic ammonium salts that serve as the sole source of nitrogen.

The growth of the organism on the acetamide agar is indicative of a positive test for acetamide utilization. During the metabolism of acetamide by a bacterium, the enzymatic action of acylamidase results in the breakdown of ammonium salts into ammonia. The ammonia thus released increases the alkalinity of the medium.

The change in pH of the medium causes the bromthymol blue indicator in the medium to turn from green to blue, indicating a positive test. In the absence of the release of ammonia, the color of the medium remains the same, indicating a negative result.

In some cases, assimilation of acetamide might result in a yellow color which could be mistaken for a positive result. However, the digestion of acetamide by deamination is limited to only a few organisms. Thus, this test is commonly performed for the differentiation of Pseudomonas aeruginosa from other non-glucose-fermenting, Gram-negative rods.

Media Used

The composition of Acetamide Agar is listed below:

1.Sodium chloride5.0
2.Magnesium sulfate0.2
3.Ammonium phosphate, monobasic1.0
4.Potassium phosphate, dibasic1.0
7.Bromothymol blue0.08
Final pH at 25°C: 6.8 ±0.2
Store at 2°C to 8°C.

Procedure of Acetamide Utilization Test

Preparation of the media

  1. In a beaker, 24.7 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of distilled or deionized water.
  2. The suspension is then heated to boiling to dissolve the medium completely.
  3. The dissolved medium is then dispensed into tubes and sterilized in an autoclave at 15 lbs pressure (121°C) for 15 minutes.
  4. Once the autoclaving process is complete, the tubes are taken out and cooled at a slanted position to a temperature of about 40-45°C. The position should be maintained in order to obtain butts of 1.5 – 2.0 cm depth.

Utilization test

  1. A well-isolated colony is taken from an 18-24 hour culture with a sterile inoculating needle.
  2. The acetamide agar tubes are inoculated by streaking the surface of the slant. The slant should be streaked back and forth with the loop or the inoculating stick.
  3. The cap of the test tubes should be left loosened to ensure adequate aeration.
  4. The tubes are then incubated aerobically at 35-37°C for up to 7 days.
  5. The test tubes should be examined daily for 4 days and again at 7 days before discarding the result as a negative.

Result Interpretation of Acetamide Utilization Test

Positive: Deamination of the acetamide, resulting in a blue color.

Negative: No color change.

Limitations of Acetamide Utilization Test

  • Growth on the slant without an accompanying color change may indicate a positive test. However, if the agar does not turn blue with further incubation, the test should be repeated with less inoculum.
  • A negative test does not rule out the identification of P. aeruginosa. Other tests should be performed for the confirmation of P. aeruginosa.
  • Only about 38% of non-pyocyanin-producing strains of P. aeruginosa produce a positive result in this test; thus, further biochemical testing should be considered for definitive identification.
  • The slant should not be stabbed as the test requires an aerobic environment.
  • The inoculums should not be taken from broth cultures as there is a chance of carryover of media with broth cultures.
  • A light inoculum should be taken to prevent the carryover of substances from previous media.

Quality Control

Positive: Pseudomonas aeruginosa (ATCC 27853)—growth; blue color

Negative: Escherichia coli (ATCC 25922)—no growth; green color

Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier