Table of Contents
- What is Acetate Utilization Test?
- Objectives of Acetate Utilization Test
- Principle of Acetate Utilization Test
- Media and Reagent used for of Acetate Utilization Test
- Procedure of Acetate Utilization Test
- Result Interpretation of Acetate Utilization Test
- Uses of Acetate Utilization Test
- Limitations of Acetate Utilization Test
What is Acetate Utilization Test?
- The acetate utilization test is a biochemical test used to identify aerobic organisms by evaluating their ability to utilize acetate present in the growth media.
- The principle of the acetate utilization test is based on an organism's ability to utilize acetate as a sole source of carbon.
- This test is similar to the acetamide utilization test and other such tests, aiding in the identification of particular groups of organisms based on their metabolic activities.
- The acetate utilization test is typically performed to differentiate Shigella from other members of the Enterobacteriaceae family.
- This test is particularly effective in differentiating Shigella from E. coli, as the majority of E. coli can utilize acetate, while most Shigella species cannot.
- The acetate utilization test also helps differentiate fermentative and oxidative groups of organisms.
- The utilization of acetate causes a change in the pH of the medium, resulting in a color change of the pH indicator used in the medium.
- The acetate utilization test is similar to the citrate utilization test, except that the medium contains acetate instead of citrate as the sole source of carbon.
Objectives of Acetate Utilization Test
- To differentiate between Gram-negative rods that are oxidase negative, nonmotile, and anaerogenic, which are likely to be either E. coli or Shigella.
- To differentiate lactose-non-fermenting, Gram-negative microorganisms from fermentative bacteria.
Principle of Acetate Utilization Test
- Acetate agar is a medium used in biochemical tests to determine the ability of an organism to utilize acetate.
- Organic acids like citrate and acetate have been widely used as a method for the differentiation of the members of the Enterobacteriaceae family.
- Most of these bacteria are capable of utilizing organic acids in the presence of organic nitrogen.
- The acetate medium contains inorganic ammonium salts as the source of nitrogen along with acetate as a source of carbon.
- The growth of the organism on the medium is indicative of a positive test for acetate utilization.
- The metabolism of acetate by the bacteria is followed by the breakdown of ammonium salts into ammonia.
- The release of ammonia increases the pH of the medium.
- The shift in pH turns the bromothymol blue indicator in the medium from green to blue.
- This medium is recommended for differentiating Shigella from Escherichia coli.
- Approximately 84% of E. coli strains utilize acetate, whereas the majority of Shigella and Proteus species are incapable of acetate utilization.
Media and Reagent used for of Acetate Utilization Test
1. Media Used
- The media used in the acetate utilization test is a growth medium containing sodium acetate as the sole source of carbon.
- The composition of the acetate agar medium is given below:
- Final pH at 25°C: 6.7 ±0.2
- Store at 2°C to 8°C.
- Acetate agar is also available commercially and might have a different composition.
The composition of commercially available acetate agar medium is given below:
- Final pH at 25°C: 5.4 ±0.2
2. Supplies Used
- Sterile inoculating loops or sticks
- Sterile pipettes
- Incubator set at 35°C
- Sterile saline
Procedure of Acetate Utilization Test
1. Preparation of media
- In a beaker, add 69.1 grams of the dehydrated powder or lab-prepared media to 1000 milliliters of distilled or deionized water.
- Heat the suspension to boiling to completely dissolve the medium.
- Dispense the dissolved medium into tubes.
- Sterilize the tubes in an autoclave at 15 lbs pressure (121°C) for 15 minutes.
- After autoclaving, remove the tubes and cool them at a slanted position to a temperature of about 40-45°C. This position should be maintained to obtain butts with a depth of 1.5 – 2.0 cm.
2. Utilization test
- A well-isolated colony from an 18-24 hour culture is taken using a sterile inoculating needle.
- Alternatively, a turbid saline suspension can be prepared using an 18 to 24 hour culture from a non-inhibitory culture plate.
- The acetamide agar tubes are inoculated by streaking the surface of the slant with either the light inoculum picked from the culture plate or a drop of the saline suspension. The slant should be streaked back and forth with the loop or the inoculating stick.
- The caps of the test tubes should be left loosened to ensure adequate aeration.
- The tubes are incubated aerobically at 35-37°C for up to 7 days. While incubation at 35-37°C for up to 5 days may be sufficient for Enterobacteriaceae, 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 negative.
Result Interpretation of Acetate Utilization Test
- A positive result in the acetate utilization test is indicated by growth and a change in color along the slant from green to intense blue.
- A negative result is indicated by no growth, no color change, with the slant remaining green throughout.
Control bacteria
- As part of quality control for the acetate utilization test, two different organisms can be used as positive and negative controls.
Control organisms are subjected to aerobic incubation at 33-37°C for 24-48 hours in the acetate utilization test, yielding the following results:
- Escherichia coli: Demonstrates acetate utilization with growth and intense blue color.
- Shigella flexneri: Shows no growth and maintains a green color, indicating acetate non-utilization.
Uses of Acetate Utilization Test
- The acetate utilization test assesses an organism's capacity to metabolize acetate as its primary carbon source.
- Additionally, it serves as a qualitative method to distinguish between fermentative and oxidative Gram-negative bacteria.
- Acetate agar is employed as a selective medium for isolating E. coli.
Limitations of Acetate Utilization Test
- Growth on the slant without a concurrent color change may suggest a positive test. However, if the agar does not turn blue upon further incubation, the test should be repeated using a smaller inoculum.
- Tests showing uncertain or equivocal results should be repeated for accuracy.
- Stabbing the slant should be avoided as the test necessitates an aerobic environment.
- Inoculums should not be taken directly from broth cultures to prevent carryover of media components.
- It is recommended to use a light inoculum to minimize the risk of carryover from previous media.
