Acetamide Utilization Test- Principle, Procedure, Results, Uses

Acetamide (CH3CONH2) is the simplest amide derived from acetic acid. It is an organic compound that can be used as a sole source of carbon by some bacteria. The ability to utilize acetamide by deamidation is a biochemical characteristic that can help in the identification and differentiation of aerobic organisms, especially Gram-negative, non-fermentative bacteria.

Deamidation is the process of breaking down acetamide into ammonia and acetic acid by the action of an enzyme called acylamidase. The ammonia released from the deamidation increases the alkalinity of the medium, which can be detected by a color change of a pH indicator.

The acetamide utilization test is performed on a medium that contains acetamide as the only carbon source and inorganic ammonium salts as the only nitrogen source. The medium also contains bromthymol blue as a pH indicator that turns from green to blue in alkaline conditions.

The acetamide utilization test is commonly used for the differentiation or identification of Pseudomonas aeruginosa from other non-glucose-fermenting, Gram-negative rods, on the basis of their ability to utilize acetamide. Pseudomonas aeruginosa is an opportunistic pathogen that can cause various infections in humans and animals. 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.

The main objective of the acetamide utilization test is to differentiate bacteria on the basis of their ability to utilize acetamide as a sole source of carbon. This test can help in the identification of aerobic organisms that can metabolize acetamide by deamidation, a process that involves the removal of an amide group from a molecule.

One of the specific applications of this test is to identify Pseudomonas aeruginosa from other non-glucose-fermenting, Gram-negative rods. P. aeruginosa is an opportunistic pathogen that can cause various infections in humans and animals. It is also known for its resistance to many antibiotics and disinfectants. Therefore, it is important to accurately identify and isolate this organism from clinical and environmental samples.

P. aeruginosa can utilize acetamide as a sole source of carbon and produce ammonia as a by-product. This results in an alkaline reaction that changes the color of the indicator in the medium from green to blue. Other non-glucose-fermenting, Gram-negative rods, such as Acinetobacter and Alcaligenes, cannot utilize acetamide and do not produce ammonia. Therefore, they do not change the color of the medium and show a negative result.

The acetamide utilization test can also be used as a qualitative test for the differentiation of Gram-negative bacteria into the fermentative and oxidative group. Fermentative bacteria are those that can break down carbohydrates anaerobically and produce acid or gas as end products. Oxidative bacteria are those that can break down carbohydrates aerobically and produce water and carbon dioxide as end products.

Acetamide is a non-carbohydrate compound that cannot be fermented by bacteria. Therefore, only oxidative bacteria can utilize acetamide as a sole source of carbon and show a positive result in this test. Fermentative bacteria cannot utilize acetamide and show a negative result in this test.

The acetamide utilization test is a simple and inexpensive method that can provide useful information for the identification and differentiation of aerobic bacteria. However, it should not be used as the sole criterion for bacterial identification, as some bacteria may show variable or ambiguous results in this test. Therefore, it should be supplemented with other biochemical tests for confirmation.

The principle of the acetamide utilization test is based on the ability of some bacteria to use acetamide as a sole source of carbon by breaking it down into ammonia and acetic acid. This process is catalyzed by the enzyme acylamidase, which is present in some Gram-negative bacteria, especially Pseudomonas aeruginosa.

The medium used for this test contains acetamide as the only carbon source and ammonium salts as the only nitrogen source. The medium also contains bromthymol blue as a pH indicator, which turns from green to blue in alkaline conditions.

When a bacterium that can utilize acetamide grows on this medium, it produces ammonia as a by-product of deamidation. The ammonia increases the pH of the medium and changes the color of the indicator from green to blue. This indicates a positive result for acetamide utilization.

On the other hand, if a bacterium cannot utilize acetamide, it will not grow on this medium or produce any color change. This indicates a negative result for acetamide utilization.

The acetamide utilization test is useful for differentiating between fermentative and oxidative bacteria, as well as for identifying P. aeruginosa from other non-glucose-fermenting, Gram-negative rods. However, this test has some limitations and should be interpreted with caution and in conjunction with other tests.

The medium used for the acetamide utilization test is acetamide agar, which contains acetamide as the sole source of carbon and inorganic ammonium salts as the sole source of nitrogen. The medium also contains bromthymol blue as a pH indicator that changes color from green to blue in alkaline conditions. The composition of acetamide agar is as follows:

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

The reagent used for the acetamide utilization test is a sterile inoculating loop or stick that is used to transfer a well-isolated colony from an 18-24 hour culture to the surface of the slant.

No other media or reagent is required for this test.

The procedure of acetamide utilization test involves the following steps:

• Preparation of the media: In a beaker, 24.7 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of distilled or deionized water. The suspension is then heated to boiling to dissolve the medium completely. The dissolved medium is then dispensed into tubes and sterilized in an autoclave at 15 lbs pressure (121°C) for 15 minutes. 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: A well-isolated colony is taken from an 18-24 hour culture with a sterile inoculating needle. 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. The cap of the test tubes should be left loosened to ensure adequate aeration. The tubes are then incubated aerobically at 35-37°C for up to 7 days. The test tubes should be examined daily for 4 days and again at 7 days before discarding the result as a negative.

The procedure is summarized in the following table:

The result of the acetamide utilization test is based on the color change of the medium after incubation. The color change is due to the pH shift caused by the release of ammonia during the deamidation of acetamide.

• A positive test is indicated by growth and change from green to blue color along the slant. This means that the organism can utilize acetamide as a sole source of carbon and produces acylamidase enzyme. An example of a positive organism is Pseudomonas aeruginosa.
• A negative test is indicated by no growth, no color change, or development of a yellow color. This means that the organism cannot utilize acetamide as a sole source of carbon or does not produce acylamidase enzyme. An example of a negative organism is Escherichia coli.

The following figure shows the acetamide utilization test results for positive and negative organisms:

Figure: Acetamide Utilization Test Results. Image Source: microbeonline.com

The acetamide utilization test has several uses in microbiology, such as:

• It is used to test the ability of an organism to utilize acetamide as a sole source of carbon. This can help in identifying the metabolic pathways and enzymes involved in the breakdown of acetamide by the organism.
• It is also used as a qualitative test for the differentiation of Gram-negative bacteria into the fermentative and oxidative group of bacteria. The fermentative group of bacteria cannot utilize acetamide as a sole source of carbon, while the oxidative group can. This can help in narrowing down the possible identification of an unknown organism.
• Acetamide agar is also used as a selective medium for the isolation of P. aeruginosa. P. aeruginosa is one of the few organisms that can utilize acetamide as a sole source of carbon and produce a blue color on the medium. This can help in isolating P. aeruginosa from other non-glucose-fermenting, Gram-negative rods that might be present in a mixed culture.
• 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. This can help in studying the nutritional requirements and preferences of an organism.

• The acetamide utilization test is not a definitive test for the identification 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 test requires an aerobic environment and a light inoculum. The slant should not be stabbed as this may create anaerobic conditions that inhibit the deamidation of acetamide. The inoculum should not be taken from broth cultures as there is a chance of carryover of substances from previous media that may interfere with the test.
• The test may give false-positive results if the organism can utilize other sources of carbon in the medium besides acetamide. For example, some organisms may be able to use citrate or succinate as carbon sources and produce alkaline products that change the color of the indicator. Therefore, it is important to use a medium that contains acetamide as the sole source of carbon and to check for growth on the slant as well as color change.
• The test may give false-negative results if the organism can utilize acetamide but does not produce enough ammonia to change the color of the indicator. This may happen if the organism has a low level of acylamidase activity or if the pH of the medium is too high to begin with. Therefore, it is important to use a fresh medium with a pH of 6.8 and to incubate the tubes for up to 7 days before discarding them as negative.

: Bailey and Scott’s Diagnostic Microbiology. Elsevier.

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