Acetamide Utilization Test- Principle, Procedure, Results, Uses

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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 their ability to utilize citrate as a sole source of carbon. This is used for the identification of organisms belonging to the Enterobacteriaceae family.

Objectives of Acetamide Utilization Test

  • To differentiate bacteria on the basis of their ability to utilize acetamide as a sole source of carbon.
  • To identify P. aeruginosa from other non-glucose-fermenting, Gram-negative rods.

Principle of Acetamide Utilization Test

  • 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 as 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 and Reagent Used for Acetamide Utilization Test

Media Used

The composition of Acetamide Agar is listed below:

S.N Ingredients Gram/liter
1. Sodium chloride 5.0
2. Magnesium sulfate 0.2
3. Ammonium phosphate, monobasic 1.0
4. Potassium phosphate, dibasic 1.0
5. Acetamide 10.0
6. Agar 15.0
7. Bromothymol blue 0.08
Final pH at 25°C: 6.8 ±0.2
Store at 2°C to 8°C.

Acetamide agar is also available commercially and might have a different composition.

S.N Ingredients Gram/liter
1. Acetamide 3.0
2. Dextrose 0.2
3. Yeast extract 0.5
4. Potassium dihydrogen phosphate 1.0
5. Phenol red 0.03
6. Bacteriological Agar 15.0
7. Sodium chloride 5.0
Final pH at 25°C: 6.8 ±0.2

Supplies or equipment used

  • Sterile inoculating loops or sticks.
  • Incubator at 35°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

Acetamide Utilization Test- Principle, Procedure and Expected Results

Figure: Acetamide Utilization Test Results. Image Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier.

  • A positive test is represented by growth and change from green to intense blue color along the slant.
  • A negative test is represented by no growth, no color change, with the slant remaining green.

Control bacteria

As a form of quality control for the acetamide utilization test, two different organisms can be taken as a positive and negative control.

Control Incubation Results
P. aeruginosa Aerobic incubation for 24-48 hours at 33-37°C. Acetamide positive (growth; blue color)


Escherichia coli Aerobic incubation for 24-48 hours at 33-37°C. Acetamide negative (no growth; green color)

Uses of Acetamide Utilization Test

  • The Acetamide utilization test is used to test the ability of an organism to utilize acetamide as a sole source of carbon.
  • It is also used as a qualitative test for the differentiation of Gram-negative bacteria into the fermentative and oxidative group of bacteria.
  • Acetamide agar is also used as a selective medium for the isolation of P. aeruginosa.
  • This medium has used a modification of Simmon Citrate agar to determine the ability of acetamide to act as a carbon source in the absence of peptone or other protein sources.

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.

References and Sources

  • Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook,–
  • Oberhofer, T. R., & Rowen, J. W. (1974). Acetamide agar for differentiation of non-fermentative bacteria. Applied Microbiology28(4), 720–721.
  • Aetamide Agar. Thermo Fisher Scientific Inc.
  • Bailey and Scott’s Diagnostic Microbiology. Elsevier.

About Author

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Anupama Sapkota

Anupama Sapkota has a bachelor’s degree (B.Sc.) in Microbiology from St. Xavier's College, Kathmandu, Nepal. She is particularly interested in studies regarding antibiotic resistance with a focus on drug discovery.

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