Acetamide Utilization Test: Principle, Procedure, and Results

Acetamide (ethanamide) is the simplest amide derived from acetic acid.  The formula of this organic compound is CH3CONH2.

Acetamide utilization test is recommended for differentiating Pseudomonas aeruginosa from other non-glucose-fermenting, gram-negative rods.  It differentiates microorganisms based on utilizing acetamide as the sole carbon source.  

Acetamide utilization test
Acetamide utilization test (A: positive, B: negative)


Acetamide broth/agar contains acetamide (CH3CONH2) as the sole carbon source and inorganic ammonium salts as the sole nitrogen source. Bacteria capable of growth on this medium produce the enzyme acylamidase which deaminates acetamide to release ammonia. The production of ammonia results in alkalinity. This shift in pH turns the bromthymol blue indicator in the medium from green to blue, causing the medium to change color from green to royal blue.

Thus, the growth and subsequent change in the blue color of the medium indicates a positive test for acetamide utilization. Assimilation of acetamide will result in a yellow color and should not be mistaken for a positive result.

Materials and Methods

  1. Sterile inoculating loops or sticks
  2. Incubator
  3. Acetamide Agar: The composition of acetamide agar is tabulated below.
IngredientsAmount (gram/liter)
Sodium chloride5.0 g
Magenesium sulfate0.2 g
Ammonium phosphate (NH4H2PO4), monobasic1.0 g
Potassium phosphate (K2HPO4), dibasic1.0 g
Acetamide10.0 g
Agar15.0 g
Bromothymol blue0.08 g
Final pH: 6.8 

Note: Acetamide broth can be used for acetamide utilization test. 

4. Test organisms

  • Isolated colonies of fluorescent pigment-producing, non-glucose-fermenting, gram-negative rods that are suggestive of Pseudomonas aeruginosa.
  • Unusual non-glucose-fermenting, gram-negative rods, as part of the identification.

Quality Control

Perform quality control (QC) on each new lot or media shipment before using them. Inspect agar for evidence of prior freezing, contamination, cracks, dehydration, and bubbles before using or storing them. Discard any blue tubes.

  1. Acetamide Positive: Pseudomonas aeruginosa (ATCC 27853), growth; blue color
  2. Acetamide Negative: Escherichia coli (ATCC 25922), no growth; green color


Time needed: 4 days

  1. Pick the inoculum from the center of a well-isolated colony from an 18-24 hour culture.

    Do not inoculate from a broth culture because the growth will be too heavy, and the carryover of media may result in false positive reactions.  

  2. Inoculate acetamide slant with a needle by streaking the slant back and forth.

    Do not stab the slant since the test requires an aerobic environment.

  3. Place cap loosely on the tube.

  4. Incubate aerobically at 35°C to 37°C for up to 4 days.

    Observe a color change from green to blue along the slant.

  5. If equivocal, re-incubate the slant for two additional days and observe the color change.

    Repeat test with equivocal results.

Expected Results

  • Positive: Growth is seen in the slant, and the color of the slant changes from green to intense blue
  • Negative: No growth, no color change, and the slant remain green.

Delftia (Comamonas) acidovorans, P. aeruginosa, and Alcaligenes faecalis can perform acetamide deamination. A combination of fluorescent pigment, acetamide deamination, and positive oxidase test results is sufficient to identify Pseudomonas aeruginosa.


  • Growth without a color change may indicate a positive test result. Repeat the test with less inoculum if further incubation results in no color change.
  • A negative test does not rule out the identification of P. aeruginosa, and 6% of Pseudomonas fluorescens organisms have been reported to give a false positive reaction. Other fluorescent Pseudomonas spp. do not give positive reactions.

References and further readings

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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