Acetamide Utilization Test – An overview

Definition

The Acetamide Utilization Test is a biochemical test used in microbiology to determine the ability of microorganisms to utilize acetamide for bacterial growth and metabolism through the process of deamidation (an important metabolic pathway involving the conversion of acetamide). This biochemical test is specifically performed for the identification of aerobic microorganisms.

Principal

• Acetamide (a simple organic compound containing carbon, hydrogen, and nitrogen) serves as the sole source of carbon and nitrogen for microorganisms.
• Microorganisms utilize acetamide through the process of deamidation, the enzymatic process occurs in the presence of acylamidase enzyme.
• This test is used to differentiate between two major groups of microorganisms: the fermentative and oxidative categories.
• It is mainly used for identifying Gram-negative, non-fermentative bacteria such as Pseudomonas aeruginosa, based on their ability to metabolize acetamide.
• Acetamide agar is used for this biochemical test.
• The medium only has acetamide for carbon and inorganic ammonium salts for nitrogen. When the bacterium uses acetamide through acylamidase, it turns ammonium salts into ammonia, making things more basic.
• As a result, the bromthymol blue indicator in the medium changes its color from green to blue, indicating a positive test result.

Media and Reagents used for Acetamide Utilization Test:

Media:

• Acetamide Agar:

 The primary medium for the Acetamide Utilization Test is Acetamide Agar.

Composition of Acetamide agar:

Sodium chloride(NaCl)	(5 g)
Ammonium dihydrogen phosphate (NH4H2PO4)	(1g)
Acetamide	(10g)
Agar	(15g)
Bromthymol blue indicator	(0.8 g) per 1000 mL
Magnesium sulphate	(0.2g)

• Acetamide: As the primary substrate, serving as the sole source of carbon.
• Ammonium salts: The sole source of nitrogen.

 It’s important to note that the specific formulations of media may vary based on laboratory protocols and the exact requirements of the Acetamide Utilization Test.

Reagent used in Acetamide Utilization Test:

• Bromthymol Blue Indicator:

This pH indicator is important for detecting changes in the acidity or alkalinity of the medium. In the presence of acetamide-utilizing microorganisms, the enzymatic conversion of ammonium salts to ammonia results an increase in alkalinity, causing a change in pH. Bromthymol Blue changes color from green to blue in response to this pH change, indicating a positive test result.

Materials required for Acetamide Utilization Test:

• Acetamide utilization test medium (made following laboratory protocol)
• Inoculating loop or needle
• Culture of test organisms
• Sterilized Petri dishes or test tubes.
• Incubator set at 35°C.

Preparation of Acetamide Agar Medium:

1. Weighing and Mixing:
2. Measure 24.7 grams of laboratory-prepared medium and place it in a clean beaker.
3. Add 1000 milliliters of distilled water to the beaker containing the medium.
4. Dissolving Medium:
5. Heat the suspension, bringing it to a boil to ensure complete dissolution of the medium. Stir the mixture to facilitate dissolution.
6. Pouring into Tubes:
7. Once the medium is completely dissolved, carefully pour it into tubes.
8. Autoclaving:
9. Sterilize the tubes containing the medium by autoclaving at 121°C for 15 minutes under 15 lbs pressure.
10. Cooling:
11. After the autoclaving process, remove the tubes from the autoclave and allow them to cool. Place the tubes in a tilted position to achieve butt (depths of 1.5–2.0 cm).
12. Temperature Control:
13. Cool the tubes at temperature of approximately 40-45°C.

Protocol for Acetamide Utilization Test:

Inoculation:

• Start with a pure culture of the test organism.
• Using a sterile loop or needle, transfer a small amount of the pure culture onto the surface of the acetamide utilization test medium.
• Ensure even distribution by streaking the inoculum across the entire surface of the medium.

Note: Avoid using a broth culture to prevent excessive growth.

Incubation:

• Place the inoculated acetamide utilization test medium in the incubator set at the recommended temperature for the specific test organism.
• The duration of incubation may vary depending on the microorganism under examination, typically ranging from 24 to 48 hours.

Observation:

• After the designated incubation period, carefully examine the medium for visible signs of growth, such as turbidity, colonies, or any noticeable color changes.
• Note any changes observed and record the time spent during the incubation.

Result Interpretation:

• Positive Result: The deamidation of acetamide leads to the production of a distinctive blue color in the medium.
• Negative Result: No color change occurs indicating the negative result.

 Advantages of Acetamide Utilization Test:

1. Metabolic Profiling:
2. The test provides insights into the metabolic capabilities of microorganisms. Understanding how bacteria interact with acetamide contributes to a more comprehensive profile of their metabolic pathways, aiding in the characterization of microbial physiology.
3. Drug Targeting:
4. The metabolic information obtained from the Acetamide Utilization Test can be valuable in drug development. It helps researchers identify specific metabolic pathways that could serve as potential targets for antimicrobial agents, aiding in the design of more effective therapeutic interventions.
5. Epidemiological Studies:
6. The test contributes to epidemiological studies by providing data on the metabolic diversity of microorganisms in different environments. This information is crucial for tracking the prevalence and distribution of specific bacterial strains.
7. Environmental Microbiology:
8. In microbial ecology, the test aids in understanding how microorganisms interact with acetamide in various environments. This knowledge contributes to insights into nutrient cycling, microbial diversity, and ecological processes, enhancing our understanding of the role of microorganisms in ecosystems.
9. Biotechnological Applications:
10. The Acetamide Utilization Test finds relevance in biotechnological processes. It allows researchers and industries to optimize microbial strains for acetamide utilization, leading to increased efficiency in bioconversion processes and sustainable production practices.

Limitation in Acetamide Utilization Test

Limitation of Growth as Indicator:

• Observation: Growth without color change may yield a false positive.
• Implication: A positive test result solely based on growth might not accurately indicate the presence of the desired characteristics.
• Action: If no color change occurs upon continued incubation, it is recommended to repeat the test with a reduced inoculum to ensure reliable results.

• Acetamide Hydrolysis for Pseudomonas Identification: Acetamide hydrolysis alone may not be a reliable method for identifying Pseudomonas bacteria producing pyocyanin. Other species might also exhibit acetamide hydrolysis, leading to potential misidentification. Complementary tests specific to Pseudomonas and pyocyanin production should be employed for accurate characterization.
• Limited Sensitivity of Acetamide Agar for P. aeruginosa Strains: Only 38% of non-pyocyanin-producing P. aeruginosa strains are expected to test positive in Acetamide Agar. This suggests that the test may have a relatively low sensitivity for identifying certain strains. To enhance accuracy, additional biochemical testing is recommended. Depending solely on this method may result in false negatives and incomplete identification.
• Need for Additional Biochemical Testing: The requirement for additional biochemical testing indicates that the initial test may not provide sufficient specificity. Relying solely on a single test, especially one with a lower sensitivity, may lead to inconclusive or inaccurate identifications. Multiple tests and a comprehensive approach are necessary for a more reliable identification process.

References and sources:

• PRESCOTT’S MICROBIOLOGY, TENTH EDITION. / Joanne M. Willey, Hofstra University, Linda M. Sherwood, Montana State University, Christopher J. Woolverton, Kent State University.—Tenth edition.
• https://www.sciencedirect.com/science/article/abs/pii/S0196439983800154
• https://byjus.com/biology/classification-of-culture-media/
• Cappuccino J.G. and Sherman N. 2008. Microbiology: A Laboratory Manual, 8th ed. Pearson Benjamin Cummings, San Francisco, CA, USA.
• Tortora, G. J., Funke, B. R., & Case, C. L. (2021). Microbiology: An introduction. Pearson Education Limited..