Table of Contents
- What is Acetamide Utilization Test?
- Objectives of Acetamide Utilization Test
- Principle of Acetamide Utilization Test
- Media and Reagent Used for Acetamide Utilization Test
- Procedure of Acetamide Utilization Test
- Result Interpretation of Acetamide Utilization Test
- Uses of Acetamide Utilization Test
- Limitations of Acetamide Utilization Test
- References
What is the Acetamide Utilization Test?
The Acetamide Utilization Test is a biochemical test used to identify aerobic bacteria based on their ability to utilize acetamide. This process occurs through deamidation, facilitated by the enzyme acylamidase. The test helps distinguish fermentative and oxidative bacteria, particularly differentiating Gram-negative, non-fermentative bacteria like Pseudomonas aeruginosa by evaluating their ability to metabolize acetamide as a sole carbon source.
This test is similar to the Citrate Utilization Test, which identifies bacteria capable of using citrate as the only carbon source, primarily for distinguishing members of the Enterobacteriaceae family.
Objectives of the Acetamide Utilization Test
- To differentiate bacteria based on their ability to utilize acetamide as a sole carbon source.
- To identify Pseudomonas aeruginosa from other non-glucose-fermenting, Gram-negative bacteria.
Principle of the Acetamide Utilization Test
Acetamide agar is used to determine whether an organism can utilize acetamide as its sole carbon source through deamidation. This medium contains acetamide and inorganic ammonium salts, serving as the only nitrogen source. Growth on this medium indicates a positive test result.
When acetamide is metabolized, acylamidase breaks it down into ammonia, increasing the medium’s alkalinity. This pH shift changes the bromothymol blue indicator from green to blue, indicating a positive result. If no ammonia is produced, the medium remains green, signifying a negative result. In rare cases, acetamide assimilation may cause a yellow coloration, which should not be misinterpreted as a positive result.
This test is primarily used for differentiating Pseudomonas aeruginosa from other non-glucose-fermenting Gram-negative bacteria.
Media and Reagents Used for Acetamide Utilization Test
Media Composition:
Standard Acetamide Agar:
Alternative Commercial Medium:
Required Supplies and Equipment:
- Sterile inoculating loops or sticks
- Incubator set at 35°C
Procedure of the Acetamide Utilization Test
Preparation of Media:
- Dissolve 24.7 g of dehydrated or laboratory-prepared media in 1000 mL of distilled or deionized water.
- Heat to boiling until completely dissolved.
- Dispense into test tubes and autoclave at 121°C for 15 minutes.
- After sterilization, cool tubes in a slanted position (40-45°C) to form a slant with a 1.5-2.0 cm deep butt.
Utilization Test:
- Select a well-isolated colony from an 18-24 hour culture using a sterile inoculating needle.
- Inoculate the acetamide agar slant by streaking the surface back and forth.
- Loosen the test tube caps to allow proper aeration.
- Incubate aerobically at 35-37°C for up to 7 days.
- Examine the tubes daily for 4 days, with a final observation on day 7 before discarding a negative result.
Result Interpretation of Acetamide Utilization Test
| Positive | Growth with a color change from green to intense blue. |
| Negative | No growth, no color change; medium remains green. |
Control Bacteria:
Uses of the Acetamide Utilization Test
- To assess an organism’s ability to utilize acetamide as a sole carbon source.
- To differentiate Gram-negative bacteria into fermentative and oxidative groups.
- As a selective medium for isolating Pseudomonas aeruginosa.
- As a modified version of Simmons Citrate Agar to evaluate acetamide metabolism in the absence of peptone or protein sources.
Limitations of the Acetamide Utilization Test
- Growth without color change may still indicate a positive test; if no blue color appears with further incubation, repeat with a lighter inoculum.
- A negative result does not definitively rule out Pseudomonas aeruginosa. Further biochemical tests are necessary for confirmation.
- Only about 38% of non-pyocyanin-producing Pseudomonas aeruginosa strains test positive; additional biochemical analysis is recommended.
- The slant should not be stabbed, as the test requires an aerobic environment.
- Inoculum from broth cultures should be avoided to prevent carryover contamination.
- Use a light inoculum to prevent residual media effects from previous cultures.
References
- Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook, 3.17.1.1–3.17.48.3. DOI:10.1128/9781555818814.ch3.17.1
- Oberhofer, T. R., & Rowen, J. W. (1974). Acetamide agar for differentiation of non-fermentative bacteria. Applied Microbiology, 28(4), 720–721.
- Acetamide Agar. Thermo Fisher Scientific Inc.
- Bailey and Scott’s Diagnostic Microbiology. Elsevier.
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