Acetate Utilization Test- Principle, Procedure, Results, Uses

Acetate utilization test is a biochemical test that determines the ability of an organism to use acetate as the sole source of carbon. Acetate is a simple organic acid that can be metabolized by some bacteria in the presence of inorganic nitrogen. The test is based on the principle that the utilization of acetate by the bacteria results in the production of ammonia, which raises the pH of the medium and changes the color of the pH indicator from green to blue.

The acetate utilization test has two main objectives:

• To differentiate between Gram-negative rods that are oxidase negative, nonmotile, and anaerogenic, which are likely to be either E. coli or Shigella. This test is particularly effective in the differentiation of Shigella from E. coli as the majority of E. coli can utilize acetate while most of the species of Shigella cannot utilize acetate.
• To differentiate lactose-non-fermenting, Gram-negative microorganisms from fermentative bacteria. This test is also one of the methods for the differentiation of the fermentative and oxidative group of organisms.

The acetate utilization test is based on the ability of an organism to utilize acetate as a sole source of carbon. The utilization of acetate causes a change in the pH of the medium, which then results in the change of the color of the pH indicator used in the medium. The acetate utilization test is similar to citrate utilization test or citrate test, except that the medium has acetate instead of citrate as the sole source of carbon.

The acetate utilization test is useful for the identification and characterization of aerobic organisms by evaluating their metabolic activities. It is also used as a selective medium for the isolation of E. coli.

The principle of the acetate utilization test is based on the ability of an organism to use acetate as a sole source of carbon. Acetate is an organic acid that can be metabolized by some bacteria in the presence of organic nitrogen. The acetate utilization test is similar to the citrate utilization test, except that the medium has acetate instead of citrate as the sole carbon source.

The medium used for the acetate utilization test is acetate agar, which contains sodium acetate as the carbon source and ammonium salts as the nitrogen source. The medium also contains bromthymol blue as a pH indicator, which turns from green to blue in alkaline conditions.

When an organism that can utilize acetate grows on the medium, it breaks down acetate into acetyl-CoA, which then enters the tricarboxylic acid cycle (TCA cycle) or the glyoxylate cycle. The TCA cycle or the glyoxylate cycle produces carbon dioxide and water as end products. The metabolism of acetate also releases ammonia from the ammonium salts in the medium. The ammonia raises the pH of the medium, causing the bromthymol blue indicator to change from green to blue. This indicates a positive test for acetate utilization.

On the other hand, when an organism that cannot utilize acetate grows on the medium, it does not break down acetate and relies on other sources of carbon and energy. The organism does not produce ammonia and does not affect the pH of the medium. The bromthymol blue indicator remains green, indicating a negative test for acetate utilization.

The acetate utilization test is useful for differentiating between some members of the Enterobacteriaceae family, such as Shigella and Escherichia coli. Most strains of E. coli can utilize acetate, while most strains of Shigella cannot. The test can also help to distinguish between fermentative and oxidative bacteria, as fermentative bacteria usually cannot utilize acetate.

The media and reagents used for the acetate utilization test are as follows:

• Acetate agar: This is the growth medium containing sodium acetate as the sole source of carbon. The composition of the acetate agar medium is given below:

The pH of the medium is adjusted to 6.8 ± 0.2 at 25°C.

• Sterile inoculating loops or sticks: These are used to transfer a well-isolated colony from an 18-24 hour culture or a drop of turbid saline suspension to the surface of the slant.
• Sterile pipettes: These are used to prepare a turbid saline suspension by using an 18- to 24-h culture from a noninhibitory culture plate.
• Incubator at 35°C: This is used to incubate the inoculated tubes aerobically for up to 7 days.
• Sterile saline: This is used to prepare a turbid suspension of the test organism if a colony is not available.

The procedure of acetate utilization test involves the following steps:

1. Preparation of media: In a beaker, 69.1 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.
2. Utilization test: A well-isolated colony is taken from an 18-24 hour culture with a sterile inoculating needle. Alternatively, a turbid saline suspension can be prepared by using an 18- to 24-h culture from a noninhibitory culture plate. The acetate agar tubes are inoculated by streaking the surface of the slant with either the light inoculum picked from the culture plate or with a drop of the saline suspension. 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. Incubation at 35-37 °C for up to 5 days insufficient for Enterobacteriaceae but incubation at 30°C for 7 days is recommended for nonfermenting, Gram-negative rods. The test tubes should be examined daily for 4 days and again at 7 days before discarding the result as a negative.

The result of the acetate utilization test is based on the observation of the growth and color change of the medium after incubation. The growth indicates the ability of the organism to utilize acetate as a sole source of carbon, while the color change indicates the change in pH due to the release of ammonia from the breakdown of ammonium salts.

• A positive test is represented by growth and change from green to intense blue color along the slant. This means that the organism can utilize acetate and produce ammonia, which raises the pH of the medium and turns the bromthymol blue indicator from green to blue. Examples of organisms that give a positive test are Escherichia coli, Pseudomonas aeruginosa, and Alcaligenes faecalis.
• A negative test is represented by no growth, no color change, with the slant remaining green. This means that the organism cannot utilize acetate and does not produce ammonia, which leaves the pH of the medium unchanged and keeps the bromthymol blue indicator green. Examples of organisms that give a negative test are Shigella spp., Salmonella spp., and Proteus spp.

The following table summarizes the result interpretation of acetate utilization test:

Figure: Acetate Utilization Test Results. Image Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier.

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

• It is used to test the ability of an organism to utilize acetate as a sole source of carbon. This can help in the identification of aerobic organisms that can metabolize acetate and produce ammonia, which increases the pH of the medium and changes the color of the indicator.
• It is also used as a qualitative test for the differentiation of Gram-negative bacteria into the fermentative and oxidative group of bacteria. Fermentative bacteria cannot utilize acetate and remain green on the medium, while oxidative bacteria can utilize acetate and turn the medium blue.
• Acetate agar is also used as a selective medium for the isolation of E. coli. E. coli can utilize acetate and grow on the medium, while most other bacteria cannot grow on acetate agar. This can help in the detection and enumeration of E. coli in water samples, food products, or clinical specimens.
• It is also used as a differential test for the identification of Shigella from other members of the Enterobacteriaceae family. Shigella cannot utilize acetate and remain green on the medium, while most other Enterobacteriaceae can utilize acetate and turn the medium blue. This can help in the diagnosis of shigellosis, a type of bacterial dysentery caused by Shigella species.

The acetate utilization test is a simple and inexpensive test that can provide useful information about the metabolic capabilities and taxonomic relationships of bacteria. It can also help in the detection and identification of important pathogens such as E. coli and Shigella.

• Growth on the slant without an accompanying color change may indicate a positive test. However, if the agar does not turn blue on further incubation, the test should be repeated with less inoculum.
• Tests with equivocal results should be repeated.
• The slant should not be stabbed as the test requires an aerobic environment.
• The inoculums should not be taken from broth cultures as there is a chance of carryover of media with broth cultures.
• A light inoculum should be taken to prevent the carryover of substances from previous media.
• Some strains of E. coli may not utilize acetate and some strains of Shigella may utilize acetate, which can lead to false-negative and false-positive results respectively.
• The acetate utilization test may not be sufficient to differentiate between some closely related species of bacteria, such as Shigella sonnei and Escherichia coli O157:H7, which both have similar biochemical profiles and can utilize acetate.
• The acetate utilization test may not be reliable for some nonfermenting Gram-negative rods, such as Pseudomonas aeruginosa and Acinetobacter baumannii, which can grow on acetate agar but do not produce a color change.

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