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Introduction of Acetamide Utilization Test

Acetamide (ethanamide) is the simplest amide derived from acetic acid. The chemical formula of this organic compound is CH3CONH2. An acetamide utilization test is recommended for differentiating Pseudomonas aeruginosa from other non-glucose-fermenting, Gram-negative bacilli/rods. It differentiates bacteria based on utilizing acetamide as the sole carbon source.

Composition of Acetamide Agar

Ingredients   Amount  (gram/liter)

Sodium chloride: 5.0

Magnesium sulfate: 0.2

Ammonium phosphate (NH4H2PO4), monobasic: 1.0

Potassium phosphate (K2HPO4), dibasic:  1.0

Acetamide:  10.0

Agar: 15.0

Bromothymol blue: 0.08

Distilled water (D/W): 1000 ml

Final pH: 6.8

Principle of  Acetamide Utilization Test

Acetamide agar contains acetamide as the sole carbon source while inorganic ammonium salts as the sole nitrogen source. Organism capable of growing on this medium produce the enzyme, acylamidase that deaminates acetamide to release ammonia. The production of ammonia results  pH shif i.e. alkalinity that turns the bromthymol blue indicator in the medium from green to blue, creating the medium to change color from green to royal blue. This type of growth and subsequent change in the blue color of the medium denotes a positive test for acetamide utilization.

Test Requirements

• Sterile inoculating wire or loop
• Bunsen burner
• Test organisms (Unusual non-glucose-fermenting, gram-negative rods, as part of the identification)
• Acetamide Agar
• Incubator
• Quality Control (QC) Strains- Positive Control (PC): Pseudomonas aeruginosa (ATCC 27853) and Negative Control (NC): Escherichia coli (ATCC 25922)

Test Procedure

1. Take the inoculum from the center of a well-isolated colony from an 18-24 hour  old culture.
2. Inoculation from a broth culture is prohibited  because the growth will be too heavy, and the carryover of medium may result in false positive reactions.
3. Inoculate the acetamide slant with a incoluating wire by streaking the slant back and forth. Stabbing  the slant is not allowed since the test requires an aerobic environment.
4. Place the cap loosely on the tube and incubate aerobically at 35°C to 37°C for up to 4 days.
5. Observe  the color change from green to blue along the slant.
6. If equivocal, re-incubate the slant for two additional days and observe the color change.
7. The equivocal results should be repeated.

Result ans Interpretation of Acetamide Assay

Acetamide Utilization Test Positive: Growth is seen in the slant, and the color of the slant changes from green to intense blue
Acetamide Utilization Test Negative: No growth, no color change, and the slant remain green.
QC Strains- Pseudomonas aeruginosa (ATCC 27853), growth; blue color-Test Positive while E. coli (ATCC 25922), no growth; green color-Test Negative

Limitations

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

Keynotes

• Isolated colonies of fluorescent pigment-producing, non-glucose-fermenting, gram-negative bacilli or rods that are suggestive of Pseudomonas aeruginosa.
• Perform quality control (QC) on each new lot  preparation or media shipment before using them. Inspect  the test  agar for evidence of prior freezing, contamination, cracks, dehydration, and bubbles prior to  using or storing them. Discard, if there is any blue tubes.
• Assimilation of acetamide will result in a yellow color and should not be mistaken for a positive result.
• Delftia  acidovorans, Pseudomonas aeruginosa, and Alcaligenes faecalis can perform acetamide deamination. A combination of fluorescent pigment, acetamide deamination, and positive oxidase test findings  are sufficient to identify Pseudomonas aeruginosa.

Bibliography

1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University press.
2. Clinical Microbiology Procedure Hand book, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
3. Colour Atlas and Text book of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
4. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
5. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
6.  Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
7. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.