Acetamide utilization Test – Principle, Procedure, Result, Uses

It is a biochemical test used for the identification and differentiation of non-fermentative Gram-negative bacteria. It is mainly applied for the confirmation of Pseudomonas aeruginosa. This test is based on the ability of the organism to utilize acetamide as the sole source of carbon and nitrogen. The test medium contains acetamide and a pH indicator which helps in observing the metabolic activity of the organism.

It is the process where the bacteria possessing the enzyme acylamidase (acetamidase) break down acetamide by deamination. During this process ammonia is released as a by-product. The release of ammonia results in the increase of pH of the medium making it alkaline. This alkalinity change is detected by the pH indicator present in the medium. In this step the utilization of acetamide confirms the enzymatic capability of the organism.

The positive reaction is indicated by a distinct color change of the medium. When phenol red is used the color changes from yellow-orange to purplish-red. If bromothymol blue is used the color changes from green to royal blue. No color change indicates a negative result where acetamide is not utilized. Thus the Acetamide Utilization Test is helpful in routine laboratory identification of non-fermentative bacteria.

Objectives of Acetamide Utilization Test

• To differentiate non-fermentative Gram-negative bacteria on the basis of acetamide utilization.
• To identify and confirm Pseudomonas aeruginosa in routine laboratory diagnosis.
• To distinguish Pseudomonas aeruginosa from other non-glucose fermenting Gram-negative rods.
• To determine the ability of organism to produce acylamidase enzyme for deamination of acetamide.
• To differentiate metabolic groups of Gram-negative bacteria based on oxidative utilization pattern.

Principle of Acetamide Utilization Test

It is a biochemical test based on the ability of certain non-fermentative Gram-negative bacteria to utilize acetamide as the sole source of carbon and nitrogen. This test depends on the presence of a specific enzyme known as acylamidase (acetamidase) in the test organism. The enzyme is responsible for the deamination of acetamide present in the medium.

In this process acetamide is hydrolyzed by the enzyme resulting in the formation of ammonia and acetic acid. The ammonia released during the reaction leads to an increase in the alkalinity of the medium. As the pH rises the pH indicator incorporated in the agar responds to this change. This is referred to as the basic principle of the test.

The alkaline shift in the medium produces a visible color change indicating a positive reaction. When phenol red is used the medium changes from yellow-orange to purplish-red and when bromothymol blue is used the color changes from green to blue. If the organism lacks the enzyme acetamidase acetamide is not utilized and there is no growth with no color change in the medium.

Media, Reagents, and Supplies

• Acetamide agar medium containing acetamide as the sole source of carbon and nitrogen.
• pH indicator such as phenol red or bromothymol blue incorporated in the medium.
• Mineral salts like sodium chloride magnesium sulphate dipotassium phosphate and monopotassium phosphate for growth support.
• Distilled or deionized water for preparation of the medium.
• Test tubes for dispensing the medium usually prepared as slants.
• Sterile inoculating loop or needle for transferring the bacterial culture.
• Autoclave for sterilization of the medium at 121°C for 15 minutes.
• Incubator maintained at 35–37°C under aerobic conditions.
• Fresh pure culture of the test organism (18–24 hours old).
• Positive control organism Pseudomonas aeruginosa.
• Negative control organism such as Escherichia coli or Stenotrophomonas maltophilia.

Acetamide Agar Composition and Preparation

Composition (Phenol Red Formulation)

• Acetamide – 10.0 g
• Sodium chloride – 5.0 g
• Dipotassium hydrogen phosphate – 1.39 g
• Potassium dihydrogen phosphate – 0.73 g
• Magnesium sulphate – 0.5 g
• Phenol red – 0.012 g
• Agar – 15.0 g
• Distilled or deionized water – 1000 ml
• Final pH – 6.9 ± 0.2 to 7.0 ± 0.2 (at 25°C)

Composition (Bromothymol Blue Formulation)

• Acetamide – 10.0 g
• Sodium chloride – 5.0 g
• Monoammonium phosphate – 1.0 g
• Dipotassium phosphate – 1.0 g
• Magnesium sulphate – 0.2 g
• Bromothymol blue – 0.08 g
• Agar – 15.0 g
• Demineralized water – 1000 ml
• Final pH – 6.8 ± 0.2 (at 25°C)

Preparation

1. Required quantity of dehydrated medium is suspended in 1000 ml of distilled or deionized water.
2. The medium is heated with frequent shaking until it dissolves completely.
3. The prepared medium is dispensed into clean test tubes.
4. Sterilization is done by autoclaving at 121°C for 15 minutes.
5. After sterilization the tubes are allowed to cool in slanted position to form agar slants.

Procedure of Acetamide utilization Test 

1. A well isolated colony is selected from a fresh pure culture (18–24 hours old) grown on non inhibitory solid medium.
2. The inoculum is taken with a sterile inoculating loop or needle and care is taken not to use broth culture to avoid false positive reaction.
3. The surface of acetamide agar slant is inoculated by streaking in a zig-zag manner or by drawing the loop straight over the slant.
4. The butt of the medium is not stabbed as the utilization of acetamide is an aerobic process.
5. The cap of the tube is kept loose to allow proper aeration during incubation.
6. The inoculated tubes are incubated at 35–37°C under aerobic conditions.
7. The tubes are observed daily for growth and colour change for up to 4 days.
8. If no definite result is seen incubation is continued and observed again up to 7 days before discarding.
9. The results are recorded based on growth and colour change of the medium.

Result of Acetamide Utilization Test

Positive Result (+)

• The organism produces acylamidase enzyme and is able to deaminate acetamide.
• Deamination of acetamide results in the release of ammonia leading to increase in alkalinity of the medium.
• With bromothymol blue indicator the medium changes from green to royal blue.
• With phenol red indicator the medium changes from yellow-orange to purplish-red.
• Common positive organisms include Pseudomonas aeruginosa, Delftia acidovorans, Alcaligenes faecalis and Achromobacter xylosoxidans.

Negative Result (–)

• The organism does not possess acylamidase enzyme and cannot deaminate acetamide.
• No alkaline shift is produced in the medium.
• With bromothymol blue indicator the medium remains green.
• With phenol red indicator the medium remains yellow-orange.
• If growth occurs with yellow colour formation it is considered negative due to assimilation of acetamide without deamination.
• Common negative organisms include Escherichia coli, Stenotrophomonas maltophilia and most strains of Burkholderia cepacia complex.

Organisms Showing Acetamide Utilization Test Result

Acetamide Positive (+)

• Pseudomonas aeruginosa
• Delftia (Comamonas) acidovorans
• Achromobacter xylosoxidans subsp. xylosoxidans
• Alcaligenes faecalis

Acetamide Negative (–)

• Escherichia coli
• Stenotrophomonas maltophilia
• Other fluorescent Pseudomonas species (except Pseudomonas aeruginosa in most cases)

Precautions

• Broth culture should not be used for inoculation as carryover of nutrients may give false positive result.
• A light inoculum should be taken from a well isolated colony of a fresh culture.
• The butt of the slant should not be stabbed as the test requires aerobic condition.
• The cap of the tube should be kept loose during incubation to allow proper aeration.
• The test should not be discarded early and incubation may be continued up to 7 days if required.
• This test should not be used as the sole criterion for identification of Pseudomonas aeruginosa.
• Yellow colour formation indicates assimilation and should not be considered as positive result.
• Proper laboratory safety precautions should be followed while handling acetamide.

Uses of Acetamide Utilization Test

• It is used for differentiation of non-fermentative Gram-negative bacteria.
• It is recommended for identification and confirmation of Pseudomonas aeruginosa.
• It is used for detection of Pseudomonas aeruginosa from water samples.
• It helps in distinguishing Pseudomonas aeruginosa from other non-glucose fermenting Gram-negative rods.
• It is used to determine the ability of organism to utilize acetamide as sole source of carbon.
• It acts as a selective test for isolation of Pseudomonas aeruginosa from mixed cultures.
• It is applied in environmental screening and infection control studies.
• It is used as a confirmatory test when routine identification results are doubtful.

Advantages of Acetamide Utilization Test

• It helps in differentiation of non-fermentative Gram-negative bacteria.
• It is highly specific for identification of Pseudomonas aeruginosa.
• It is useful for confirmation of Pseudomonas aeruginosa isolated from water samples.
• It identifies organisms producing acylamidase enzyme for acetamide deamination.
• It allows differentiation based on utilization of acetamide as sole source of carbon and nitrogen.
• It gives clear and distinct colour change making interpretation easy.

Limitations of Acetamide Utilization Test

• Only a low percentage of apyocyanogenic strains of Pseudomonas aeruginosa gives positive result.
• Pyocyanin producing strains may interfere with colour change and make interpretation difficult.
• A negative result does not rule out Pseudomonas aeruginosa and further tests are required.
• Use of broth culture as inoculum may give false positive reaction due to ammonia production.
• Some organisms deaminate acetamide slowly and may require prolonged incubation up to 7 days.
• The test requires strict aerobic condition and stabbing of the medium may lead to false negative result.
• Growth alone does not indicate positive test as assimilation without deamination is considered negative.
• Few strains of other Pseudomonas species may give false positive reaction.